Identification of TMCO2 as an acrosome‐associated protein during rat spermiogenesis

We isolated the transmembrane and coiled‐coil domains 2 (Tmco2) gene using a polymerase chain reaction‐based subtraction technique. Tmco2 is predominantly expressed in rat testes starting from 4 weeks of age. Rat TMCO2 consists of 187 amino acids with a predicted molecular mass of 20.6 kDa. When expressed in COS7 cells, TMCO2 was found as vesicle‐like structures in the cytoplasm, whereas TMCO2ΔTM lacking the transmembrane (TM) region was found diffused in the cytoplasm. These results suggest that the TM region in TMCO2 is essential for its specificity of localization. Immunocytochemical analyzes indicated that rat TMCO2 was localized as small semiluminate bodies or cap‐like structures in the vicinity of round spermatid nuclei and as curved lines associated with nuclei of elongated spermatids and caput epididymal spermatozoa. However, it was detected in only a small part of cauda epididymal spermatozoa. Double immunolabeling of the spermatids and spermatozoa with the anti‐TMCO2 antibody and the monoclonal anti‐MN7 antibody showed that TMCO2 was predominantly associated with the inner acrosomal membrane in spermatids and caput epididymal spermatozoa. Our findings suggest that TMCO2 might be involved in the process of acrosome biogenesis, especially binding of acrosome to a nucleus, during spermiogenesis.     

Lysosome associated membrane protein 1 (Lamp1) traffics directly from the TGN to early endosomes

The precise trafficking routes followed by newly synthesized lysosomal membrane proteins after exit from the Golgi are unclear. To study these events we created a novel chimera (YAL) having a lumenal domain comprising two tyrosine sulfation motifs fused to avidin, and the transmembrane and cytoplasmic domains of lysosome associated membrane protein 1 (Lamp1). The newly synthesized protein rapidly transited from the trans- Golgi Network (TGN) to lysosomes (t(1/2) approximately 30 min after a lag of 15-20 min). However, labeled chimera was captured by biotinylated probes endocytosed for only 5 min, indicating that the initial site of entry into the endocytic pathway was early endosomes. Capture required export of YAL from the TGN, and endocytosis of the biotinylated reagent, and was essentially quantitative within 2 h of chase, suggesting that all molecules were following an identical route. There was no evidence of YAL trafficking via the cell surface. Fusion of TGN-derived vesicles with 5 min endosomes could be recapitulated in vitro, but neither late endosomes nor lysosomes could serve as acceptor compartments. This suggests that contrary to previous conclusions, most if not all newly synthesized Lamp1 traffics from the TGN to early endosomes prior to delivery to late endosomes and lysosomes.     

Unifying nomenclature for the isoforms of the lysosomal membrane protein LAMP-2

The present nomenclature of the splice variants of the lysosome-associated membrane protein type 2 (LAMP-2) is confusing. The LAMP-2a isoform is uniformly named in human, chicken, and mouse, but the LAMP-2b and LAMP-2c isoforms are switched in human as compared with mouse and chicken. We propose to change the nomenclature of the chicken and mouse b and c isoforms to agree with that currently used for the human isoforms. To avoid confusion in the literature, we further propose to adopt the use of capital letters for the updated nomenclature of all the isoforms in all three species: LAMP-2A, LAMP-2B, and LAMP-2C.     

A membrane protein,TMCO5A,has a close relationship with manchette microtubules in rat spermatids during spermiogenesis

We isolated the transmembrane and coiled‐coil domains 5A (Tmco5A) gene using polymerase chain reaction‐based subtraction technique and showed that Tmco5A was predominantly expressed in rat testes starting at 4 weeks of postnatal development. When expressed in COS7 cells, TMCO5A was found to be distributed in the endoplasmic reticulum‐nuclear membrane (ER‐NM) of cells as a membrane‐associated protein, while TMCO5AΔC lacking the transmembrane region (TM) mislocalized and diffused throughout the cytoplasm. The result suggested that TM is responsible for the retention of TMCO5A at the ER‐NM. Immunocytochemical and immunoblotting analyses indicated that TMCO5A was localized along the posterior part of the nuclei in both round and elongated rat spermatids but disappeared from epididymal spermatozoa. Double immunolabeling of isolated spermatids with the anti‐TMCO5A and the anti‐β tubulin antibodies showed that TMCO5A was always found to be closely associated with developing manchette microtubules but did not completely colocalize with them. On the other hand, we found that almost all TMCO5A colocalized with SUN4, a linker of nucleoskeleton and cytoskeleton complex protein present at the posterior part of spermatid nuclei. These data suggested that TMCO5A is located closer to the nuclei than the manchette microtubules. It is likely that TMCO5A, in association with manchette microtubules, is involved in the process of spermiogenesis.     

Ultrastructure of spermiogenesis in the American alligator,Alligator mississippiensis (Reptilia,Crocodylia, Alligatoridae)

Testicular samples were collected to describe the ultrastructure of spermiogenisis in Alligator mississipiensis (American Alligator). Spermiogenesis commences with an acrosome vesicle forming from Golgi transport vesicles. An acrosome granule forms during vesicle contact with the nucleus, and remains posterior until mid to late elongation when it diffuses uniformly throughout the acrosomal lumen. The nucleus has uniform diffuse chromatin with small indices of heterochromatin, and the condensation of DNA is granular. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. Once the acrosome has completed its development, the nucleus of the early elongating spermatid becomes associated with the cell membrane flattening the acrosome vesicle on the apical surface of the nucleus, which aids in the migration of the acrosomal shoulders laterally. One endonuclear canal is present where the perforatorium resides. A prominent longitudinal manchette is associated with the nuclei of late elongating spermatids, and less numerous circular microtubules are observed close to the acrosome complex. The microtubule doublets of the midpiece axoneme are surrounded by a layer of dense staining granular material. The mitochondria of the midpiece abut the proximal centriole resulting in a very short neck region, and possess tubular cristae internally and concentric layers of cristae superficially. A fibrous sheath surrounds only the axoneme of the principal piece. Characters not previously described during spermiogenesis in any other amniote are observed and include (1) an endoplasmic reticulum cap during early acrosome development, (2) a concentric ring of endoplasmic reticulum around the nucleus of early to middle elongating spermatids, (3) a band of endoplasmic reticulum around the acrosome complex of late developing elongate spermatids, and (4) midpiece mitochondria that have both tubular and concentric layers of cristae. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Fine structure of spermiogenesis with special reference to the spermatid morulae of the freshwater mussel Prisodon alatus (Bivalvia,Unionoidea)

Within the testicular cysts of the mussel Prisodon alatus are numerous somatic host cells described as Sertoli cells (SC), each containing a variable number of young spermatid morulae. Among them, several free spermatid morulae, spermatids, and spermatozoa were observed. Each free spermatid morula is surrounded by an external membrane. The early spermatids enclosed within the morulae have dense and homogeneous chromatin, and the cytoplasm occupies little space around the nucleus. Later, during spermiogenesis, the SC show lysis and disrupt to liberate the spermatid morulae. The membrane of the free morula is then disrupted, releasing the young spermatids. The SC disappear just after the appearance in the testis of a large number of free young spermatids. The nucleus of each free spermatid becomes gradually smaller and denser by the appearance of a granular pattern of condensed chromatin. During the maturation phase of the spermatids, the cytoplasm becomes more voluminous, and mitochondria and centrioles are more evident. Then, flagellogenesis occurs, and the nucleus gradually condenses into thicker strands. In the mature sperm, the apical zone has a disc-shaped acrosomal vesicle and the midpiece contains five mitochondria and two centrioles located at the same level. The flagellum has the common 9+2 microtubular pattern. The results are discussed with particular reference to Sertoli cells and clusters of spermatid morulae with those of species of closely related taxa in the bivalves. J. Morphol. 238:63–70, 1998. © 1998 Wiley-Liss, Inc.     

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac2,-3Gal1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.     

Characterization of rtSHSp13 gene encoding a development protein involved in vesicular traffic in spermiogenesis

A cDNA, designated as rtSH3p13, was isolated from a rat testis cDNA library. It consists of 1463 bp nuclear acids, which encodes a protein of 312 amino acids and was assigned the GenBank accession number AF227439. The deduced rtSH3p13 protein is a truncated isoform of SH3p13 as a result of mRNA alternative splicing. It is mainly expressed in the rat testis, detected in spermatids at the steps 8-19 of spermiogenesis, and found around the acrosome. During postnatal development, rtSH3p13 appears on day 18 and reaches maximum on day 60. Further experimental results suggested that rtSH3p13 forms a complex with activated epidermal growth factor receptor (EGFR) and interacts with synaptojanin I. Surprisingly, similar to SH3 domain, the V region of rtSH3p13 also inhibits endocytosis in CHO cells. Our results reveal a link between an rtSH3p13-synaptojanin-clathrin complex-mediated formation of pits and the process of spermiogenesis.     

AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways

The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19°C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro . The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.     

Complementary DNA cloning of rat spetex-1, a spermatid-expressing gene-1, encoding a 63 kDa cytoplasmic protein of elongate spermatids

We used differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene designated as spetex-1, which had an open reading frame of 1,668-length nucleotides encoding a protein of 556 amino acids. Spetex-1 mRNA was highly expressed in testis, and weekly expressed in lung, intestine, and spleen. Spetex-1 expression in the rat testes was detected first at 3 weeks in postnatal development and continued to be detected up to adulthood. A search in the databases showed that the amino acid sequence of spetex-1 was 82% identical to that of its mouse homologue found in the databases. Both rat spetex-1 and the mouse homologue contained Ser-X (X = His, Arg, or Asn) repeats in the middle portion of the proteins. In situ hybridization revealed that spetex-1 mRNA was expressed in haploid spermatids of step 7-18 within the seminiferous epithelium. Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spetex-1 protein was not expressed in spermatogonia, spermatocytes, and round spermatids in adult rat testis, but was specifically detected in the residual cytoplasm of elongate spermatids of step 15-18 as well as in residual bodies engulfed by Sertoli cells. We interpreted these data as a potential role of spetex-1 in spermatogenesis, especially in cell differentiation from late elongate spermatids to mature spermatozoa.     

Temporal expression and localization of protein farnesyltransferase during spermiogenesis and posttesticular sperm maturation in the hamster

Spermiogenesis and posttesticular sperm maturation in the epididymis are distinct developmental processes that result in a polarized spermatozoon possessing a plasma membrane partitioned into segment-specific domains of distinct composition and function. The mechanisms that specify the distribution of intracellular organelles and target proteins to restricted membrane domains are not well understood. In this study we examined the expression pattern and distribution of protein farnesyltransferase (FTase) in hamster spermatids and epididymal spermatozoa to determine if protein lipidation may represent a potential mechanism to regulate protein association with specific organelles or the plasma membrane. Round spermatids exhibited only weak immunostaining with antibody against the β-subunit of FTase, whereas elongating spermatids exhibited a high level of FTase expression that was segregated to the cytoplasmic lobe surrounding the anterior flagellum. Although FTase was released with the residual body, mature spermatids retained FTase within the midpiece and cytoplasmic droplet. In epididymal spermatozoa, FTase remained associated with the cytoplasmic droplet during its migration to the midpiece-principal piece junction; following release of the cytoplasmic droplet, no immunodetectable FTase was noted in the midpiece segment. Immunoblotting demonstrated the presence of both the α and β subunits of FTase in sperm lysates. The temporal expression pattern and restricted distribution of FTase in spermatids and epididymal spermatozoa suggest a potential role in regulating protein association with specific organelles and/or membrane domains of the mature spermatozoon. Mol. Reprod. Dev. 48:71–76, 1997. © 1997 Wiley-Liss, Inc.     

精子形成期基因转录表达的研究进展

减数分裂后, 圆形精子细胞经过一系列变态过程最终发育为成熟精子。期间, 精子细胞质逐渐丢失, 其染色质组蛋白逐渐经过渡蛋白替换为鱼精蛋白, 染色质被致密包装并高度浓缩。很多学者认为, 精子转录活性被关闭, 不存在RNA。但近些年却在精子中检测到了种类繁多的转录本, 包括精子染色质重新包装所需蛋白的转录本及一些小分子RNA等。由于精子核内组蛋白没有完全被鱼精蛋白替换, 且染色质上包含一些核酸活性敏感位点, 推测精子存在一定的转录活性, 并通过激素和表观遗传修饰等调控转录。精子中的这些RNA一部分是精子形成过程中残留下来的, 另一部分是精子细胞适时表达的。深入研究精子形成中的基因转录表达, 可增进对精子形成与成熟遗传本质的理解, 为高效利用雄性配子进行生殖控制提供理论依据。文章综述了近年来精子形成期基因转录表达的研究进展, 并提出了未来的研究方向。     

Immunoelectron microscopical detection of tubulins during spermiogenesis in phytophagous bugs (Hemiptera: Pentatomidae)

Summary

Ultrastructural and immunocytochemical studies were carried out in the tail region of spermatids and spermatozoa of the phytophagous bugs, Acrosternum aseadum and Euchistus heros. The axoneme presented a 9+9+2 microtubule pattern and bridges occurred between axonemal microtubules 1, 5, and mitochondrial derivatives. Two paracrystalline structures, embedded in an amorphous matrix, were observed in the mitochondrial derivatives. The axonemal microtubules contained alpha, acetylated and tyrosinated tubulin. Cytoplasmic microtubules contained alpha, beta and gamma tubulin. Moreover, the gamma tubulin was detected near the electron dense rod, an element associated with the centriole, suggesting that this structure may be a microtubule organizing center.     

优雅蝈螽精子形成过程中F-肌动蛋白的动态变化

为阐明F-肌动蛋白在优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl精子形成过程中的动态变化, 本研究利用微分干涉相衬技术和免疫荧光技术首次对优雅蝈螽精子形成过程中的F-肌动蛋白进行了细胞定位, 利用透射电镜技术从超微水平观察了优雅蝈螽精子顶体复合体的结构。结果显示： 精子形成早期, F-肌动蛋白富集于亚顶体区域, 形态由“球状”转变为“棒锥状”; 精子形成中期, F-肌动蛋白呈“倒Y型”分布于亚顶体区域和细胞核前端两侧; 精子形成后期, 亚顶体区域的F 肌动蛋白解聚消失, F-肌动蛋白呈“箭头状”, 仅分布于顶体复合体扩张的两翼中。F-肌动蛋白动态变化伴随着细胞核和精子头部的形态改变, F-肌动蛋白的动态装配在精子顶体复合体形态构建和细胞核的形变中起着重要的作用。本研究还发现未成熟的精子尾部有一些富含F-肌动蛋白的细胞质微滴, 与精子形成过程中多余细胞质和细胞器的外排有关。F-肌动蛋白的动态变化研究为进一步阐明细胞骨架蛋白在昆虫精子形成过程中的功能和作用机制奠定了基础。     

Fine structure of the germinating cells during the spermiogenesis of Arion rufus (Mollusca,Pulmonate Gastropoda)

Changes in spermatozoan ultrastructure have been studied during spermiogenesis of the slug Arion rufus (Gastropoda, Pulmonata, Stylommatophora). The ovotestis was investigated during the male stage, definite by the presence of spermatozoa. Some peculiar characteristics are shown by early spermatids: Around the nucleus, the nuclear envelope presents two thick layers located on opposite sides, the apical and basal plates, that will determine the antero-posterior axis of the spermatid. The chromatin, first dispersed throughout the nucleoplasm gives later on thick filaments which become attached over the inner surface of these plates. The chromatin filaments are then arranged parallel to the antero-posterior axis as the nucleus elongates. The position of the plates determines the antero-posterior axis of the spermatid. In the mature spermatozoa, the chromatin is more condensed and the nucleus presents an helical organization. The acrosome and flagellum are respectively attached externally to the center of the apical and basal plates. The acrosome consists of a membrane-bound vesicle and forms a column of homogeneous material. In the middle piece, the mitochondria have been transformed into a mitochondrial derivate by the way of a complicated metamorphosis. The axoneme is surrounded by three mitochondrial helices but only one of them contains glycogene granules. © 1996 Wiley-Liss, Inc.     

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.