THE FINE STRUCTURE OF NUCLEI DURING SPERM MATURATION IN THE LOCUST

1. In the heads of maturing sperm of a locust the chromatin becomes arranged in a highly regular manner so as to produce many parallel lines about 60 A thick in longitudinal section and contiguous polygons in transverse section. 2. This configuration appears after fixation in osmium tetroxide, formaldehyde, or acetic acid; intermediate stages in its development are illustrated. 3. These electron micrographs are interpreted to mean that, during sperm maturation, the chromatin becomes formed into sheets and then into tubes running parallel with the long axis. 4. In the mature sperm head we have been unable so far to detect this structure. This may be because the chromatin becomes so compact during the shrinkage of the nucleus which occurs during formation of the mature sperm.     

Ultrastructural and chemical study of the chromatin during spermiogenesis of the fish Scyliorhinus caniculus (L.) (author's transl)

Electron microscopic, cytochemical and biochemical techniques were applied to study structural aspects and changes in nuclear components during the spermiogenesis of Scyliorhinus caniculus. Five major stages of nuclear differentiation were recognized and characterized by variations in the organization and chemical properties of chromatin. Stage I is analogous to a somatic nucleus with heterogeneous chromatin. At the second stage, the nuclear content is dispersed but the chromatin fibers are of the same diameter as those of the stage I. The nuclear elongation begins at stage III, the DNP fibers running preferentially parallel to the long axis of the nucleus. During these early modifications of chromatin structure appear two new basic nuclear proteins (S 1 and S 2) which migrate faster than histones but typical histones remain assosciated with these nuclei. In later elongation stage (stage IV), the chromatin fibers organize in a helical form and fuse side by side giving lamellar systems which have a reticular structure. At the end of this stage, the nuclear material has become uniformly compact. These late variations in chromatin organization are parallel to the association of chromatin with new basic nuclear proteins (S 3, S 4, Z 1, Z 2 and Z 3). The cytochemical and electrophoretical properties of one of these proteins (S 4) which appears at the end of spermiogenesis are similar to those of a protamine. In stage V, the chromatin is homogeneous and the nucleus assumes a helical configuration beginning at the posterior end. The deoxyribonucleoproteins of the mature sperm show some novel chemical characters, including the appearance of a stable nuclear acidophilia with the ALFERT and GESCHWIND method and extraction with 0.25 N HCl of one of the basic protein fractions newly appeared in late spermiogenesis (Z 3), two other fractions (Z 1 and Z 2) being extracted with a more drastic procedure. The other fractions described before are no more detectable.     

Ultrastructure of spermiogenesis and the spermatozoon in Castrada cristatispina Papi, 1951 (Platyhelminthes, Rhabdocoela, Typhloplanida)

Spermiogenesis in Castrada cristatispina begins with the formation of a zone of differentiation containing two centrioles with associated striated rootlets and an intercentriolar body between them. The centrioles give rise to two parallel, free flagella of the Trepaxonemata 9 + '1' pattern, growing out in opposite directions. Spermatids undergo a latero-ventral rotation of the flagella and a subsequent disto-proximal rotation of centrioles, and a distal cytoplasmic projection appears. The former rotation involves the compression of a row of microtubules and allows the recognition of a ventral side and a dorsal side. At the end of the differentiation, the centrioles and cortical microtubules lie parallel to the sperm axis. The modifications of the intercentriolar body and the migration of the nucleus and the centrioles toward the distal projection are described. The mature spermatozoon of C. cristatispina is filiform, tapered at both ends and shares several features with the other Rhabdocoela gametes. Nevertheless, the posterior extremity is capped by an electron-dense material. A gradient between mitochondria and dense bodies exists along the sperm axis. This study has enable us a phylogenetic approach of the Rhabdocoela through a comparison of the ultrastructural features of C. cristatispina with the other Rhabdocoela taxa. We propose the disto-proximal rotation of centrioles as a synapomorphy of the Rhabdocoela.     

Spermiogenesis and the spermatozoon ultrastructure of Robphildollfusium fractum (Digenea: Gyliauchenidae), an intestinal parasite of Sarpa salpa (Pisces: Teleostei)

Spermiogenesis in Robphildollfusium fractum begins with the formation of a differentiation zone containing: two centrioles, each bearing striated rootlets, nucleus, several mitochondria and an intercentriolar body constituted by seven electron-dense layers. The two centrioles originate two free flagella growing orthogonally to the median cytoplasmic process. Later, the free flagella rotate and undergo proximodistal fusion with the median cytoplasmic process. Nuclear and mitochondrial migrations occur before this proximodistal fusion. Finally, the young spermatozoon detaches from the residual cytoplasm after the constriction of the ring of arched membranes. The spermatozoon of R. fractum exhibits two axonemes of different length of the 9 + ‘1’ trepaxonematan pattern, nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. Additionally, a shorter axoneme, which does not reach the nuclear region, the presence of an electron-dense material in the anterior spermatozoon extremity and the morphologies of both spermatozoon extremities characterize the mature sperm of R. fractum.     

STUDIES OF SPERMIOGENESIS IN A NEMATODE, NIPPOSTRONGYLUS BRASILIENSIS

The fine structure of the developing spermatids and the mature sperm of Nippostrongylus brasiliensis was investigated. Immature spermatids are found at one end of the tubelike testis, and the mature sperm at the other. The spermatid has a prominent nucleus, with the chromatin clumped at the margin. It also contains a pair of centrioles, located near the nucleus. The cytoplasm is filled with ribosomal clusters, but it lacks an organized Golgi area or endoplasmic reticulum. Besides the normal mitochondria, the spermatid has specialized mitochondrionlike inclusions with dense matrix, few broad cristae, and a crystalloid structure always facing the nucleus. As spermiogenesis proceeds, the nucleus elongates, comes to lie at one end, and later evaginates to form a separate head structure, leaving the mitochondria and other cytoplasmic organelles in a broad cytoplasmic region. The nuclear material becomes filamentous and spiral, and the centrioles come to lie at one end near the junction of the head and the cytoplasmic portion of the sperm. Microtubules are found in the cytoplasmic region extending from the tubelike nucleus. The specialized mitochondria are about eighteen in number, and are arranged in rows in staggered groups of three around the microtubules in the cytoplasmic region. The mature sperm is aflagellate and lacks an acrosome. No movement of the sperm was ever observed.     

A birefringence, electron microscopy, and histochemical survey of chromosomal structure in the sperm nuclei of an echinothurid sea urchin, Araeosoma owstoni

The mature sperm head of Araeosoma owstoni, an echinothurid sea urchin, showed positive birefringence reflecting that the overall orientation of DNA molecules was semiperpendicular toward the nuclear axis of the sperm head. Transmission electron microscopical observation of sperm in this species revealed a highly electron-dense cylindrical coil with an empty central core extending along the major axis of the sperm head. This coil had seven to eight turns along its entire length of 3.5 micron. The maximum width was 0.35 micron near the distal end of the nucleus, and the minimum width was 0.17 micron near the apical end. Lamellar substructures were also present in the sperm nucleus, appearing at the periphery of the electron-dense cylinder in a radial manner. Staining with Feulgen's reaction and acid-orcein indicated that the coil was probably composed of sperm chromosomes.     

Spermiogenesis and sperm in Mesostoma viaregginum (Plathelminthes, Rhabdocoela): an ultrastructural study to infer sperm orientation

Spermiogenesis in Mesostoma viaregginum begins with the formation of a zone of differentiation containing striated rootlets, two centrioles, and an intercentriolar body in-between. These centrioles generate two parallel free-flagella with the 9+“1” pattern of the Trepaxonemata growing out in opposite directions. Spermatid differentiation is characterised by a 90° latero-ventral rotation of flagella and a subsequent disto-proximal centriolar rotation, with a distal cytoplasmic projection. The former rotation involves the compression of a row of cortical microtubules and allows recognising a flagellar side and an aflagellar side in the late spermatid and in the mature spermatozoon. At the end of the differentiation, centrioles and microtubules lie parallel to the spermatid axis. The disto-proximal centriolar rotation is proposed as a synapomorphy for the Rhabdocoela. The modifications of the intercentriolar body during spermiogenesis and the migration of the nucleus and the centrioles towards the cytoplasmic distal projection are also described. The mature spermatozoon of M. viaregginum is filiform and tapered at both ends and presents many features found in the Rhabdocoela gametes. The nucleus disappears before the flagellar insertion and a density gradient of mitochondria is observed along the sperm axis. The anterior end of the spermatozoon of M. viaregginum is characterised by a tapering capped by a membrane expansion. This study has enabled us to describe precisely the orientation of spermatozoa in the Rhabdocoela in general: the centriolar extremity is proposed as the anterior one for the Rhabdocoela.     

SPERMIOGENESIS IN WILD TYPE AND IN A MALE STERILITY MUTANT OF DROSOPHILA MELANOGASTER

Spermiogenesis in the translocation heterozygote T (1; 2H) 25(20) y l 25/FM6 has been studied with the electron microscope and compared with that in wild type males. It appears that the genetic lesion in the male sterility mutant is associated primarily with a failure in differentiation of the head. In wild type flies, the spermatid nucleus assumes a conchoidal shape; chromatin accumulates along the convex surface. Adjacent to the concave surface a large bundle of microtubules runs parallel to the long axis of the spermatid. A single row of microtubules is juxtaposed against the convex surface of the head. As differentiation proceeds, the nucleus elongates, chromatin condenses, and the nucleus is compacted to a final diameter of about 0.3 µ. In the sterile mutant the spermatid nucleus has an irregular or wedge-shaped profile and no concavity is formed, nor is the bundle of microtubules observed. The row of microtubules, however, is usually present around the periphery. The change from lysine-rich to arginine-rich histone in mature wild type sperm does not occur in the sterile male. The substructure of the axial filament and mitochondrial derivatives, however, are similar to those in wild type.     

SPERMIOGENESIS IN THE PULMONATE SNAIL, EUHADRA HICKONIS II. STRUCTURAL CHANGES OF THE NUCLEUS

In accordance with the characteristic shape of the nucleus and degree of condensation of the nuclear substance, spermiogenesis in Euhadra hickonis can be roughly divided into four stages. The chromatin in the highly polymorphic nucleus of the first stage, early spermatid, forms relatively thick (ca. 50 nm) fibrils which associate here and there into irregular clumps. In the next stage, the spermatid nucleus becomes conspicuously spherical, its contents appear more finely homogeneous and the irregular clumps of chromatin are few. In the third stage, the nucleus gradually takes on an ellipsoidal shape as the antero-posterior axis shortens. The anterior part of its envelope becomes structurally modified in preparation for the adherence to it of the developing acrosome, and an implantation fossa forms posteriorly at the center of a second area where the nuclear envelope has been modified. The diameter of the chromatin fibrils again increases and those near the implantation fossa become oriented perpendicular to the nuclear envelope.
As the nucleus elongates in the fourth stage, a concentric sheath of microtubules closely surrounds it. These appear to depolymerize as the nuclear elongation proceeds, so that they are no longer present in the head region of the mature spermatozoon. The diameter of the chromatin fibrils increases to about 10 nm and they become oriented parallel to the long axis of the cell. With the decrease in the nuclear volume the fibrils unite laterally to form longitudinal sheets, and these finally merge in the mature spermatozoon into a mass of very dense chromatin without perceptible internal structure.     

Fine structure and development of the cuticle of Intoshia variabili (Orthonectida)

The body of free-swiming mature Intoshia variabili (Orthonectida) is covered by a thin cuticle, 0.3 μm thick. The cuticle is formed at the time when the orthonectid embryos develop in the plasmodium. The process of cuticle formation begins just after the first cilia begin to appear at the surface of the ciliated cells. At first, small extensions of the cell membrane appear at the surface of the cell, more or less parallel to the cell surface. As they develop futher, they stand up, and amorphic material begins to appear between them. The extensions then become microvilli and obtain their final shape, with a small subdistal swelling and a narrower distal part. They are situated very regularly on the surface of the cell. After the microvilli have obtained their final form, material between them begins to get its final structure typical of the adult form. During the period when the mature orthonectid begins to leave the plasmodium and emerge from the host, the regular microvilli begin to disappear, and only small irregular extensions are present under the cuticle on the surface of the cell. During the process of cuticle formation a large amount of smooth endoplasmic reticulum develops in the cells, but once the cuticle is formed it gradually disappears.     

The fine structure of the sperm bundles of the coccid,Aspidiotus perniciosus Cumst., and sperm movement

The mature sperm of A. perniciosus are organized into bundles, about 350 μm long by 9–10 μm wide. Each bundle contains 32 sperm enclosed by a common sheath. The sperm contains an elongated ‘central core’, representing nuclear material, surrounded by a spiral microtubular sheath and cytoplasm. The electron-dense nuclear material is localized in the more pointed half of the sperm. The spiral microtubular sheath is composed of 30— 100 microtubules (depending on the cross-sectional level), situated parallel to the longitudinal axis of the sperm. On the basis of this ultrastructural organization, the motility of the sperm and sperm bundle as a whole is discussed. The sperm of A. perniciosus provide strong evidence that the microtubules arranged asymmetrically represent the elements directly involved in sperm motility.     

ELECTRON MICROSCOPE STUDIES ON SPERM DIFFERENTIATION IN MARINE ANNELID WORMS. I. SPERM FORMATION IN IKEDOSOMA GOGOSHIMENSE

The ultrastructur of spermatozoa and the changes through which they are differentiated during sperm formation in an echiuroid were observed under the electron microscope. Many spermatids are connected to one central cytoplasmic mass and the sperm differentiation proceeds synchronously in one sperm-ball. Dense plate-like structures appear in the cytoplasm of early spermatids and disappear soon. In the process of nuclear condensation, many electron-dense aggregates appear in homogeneously textured chromonema and the aggregates are packed together to form a uniformly dense nucleus. Near the centriole at the opposite side from the central mass, the mitochondria fuse together to form one large middle-piece mitochondrion and the acrosomal vesicle is formed from the Golgi-complex. The differentiating acrosome in the late spermatid moves to the anterior tip of the head. In the completed acrosome, a flocculent substance accumulates in the conspicuously expanded invaginated pocket of the acrosomal vesicle and two kinds of material of different electron density fill the inside of the acrosomal vesicle. The spermatozoa remain connected to the central mass at the lateral side of the head until they become fully mature and are packed into the nephridia before spawning.     

Failure of differentiation of the nuclear-perinuclear skeletal complex in the round-headed human spermatozoa

Acrosomeless round-headed spermatozoa from three men were studied under electron microscopy and indirect immunofluorescene microscopy using the anti-calicin antibody that recognizes a basic protein of the sperm perinuclear theca (Longo et al., 1987). Electron microscopy revealed the existence of anomalies of the nuclear envelope, the nuclear matrix underlying the nuclear envelope, and the perinuclear layer. The absence of sperm labeling with the anti-calicin antibody confirmed that the formation of the perinuclear theca was impaired. Data obtained from both mature spermatozoa and ejaculated spermatids suggest that i) round-headed sperm head anomalies result from a failure of differentiation of the sperm-specific skeletal complex related to the nucleus, and ii) the acrosome spreading over the nucleus, the nuclear elongation and the post-acrosomal sheath formation are dependent on such nuclear-perinuclear differentiations. In contrast, chromatin condensation, cytokinesis and some events of the acrosomal shaping appear not to depend on those nuclear-related differentiations. The possible processes allowing the maintenance of the sperm head structures and their subsequent morphogenesis are discussed.     

Ultrastructural differentiation of sperm tail region in Diplometopon zarudnyi (an amphisbaenian reptile)

Diplometopon zarudnyi, a worm lizard belongs to amphisbaenia under trogonophidae family. This species exists in limited areas of the Arabian Peninsula and is an oscillating digger found in sub-surface soils. The present study aimed to investigate the sperm tail differentiation in D. zarudnyi. Ten male adults of D. zarudnyi were collected from Riyadh during April–May 2011. To study the sperm tail at the ultrastructural level the testes were fixed in 3% glutaraldehyde, than post fixed in 1% osmium tetaroxide followed by dehydration in ethanol grades; samples were cleared in propylene oxide and embedded in resin. Tail formation begins by the moving of centrioles and mitochondria towards the posterior pole of sperm head. Simultaneously many microtubules of the midpiece axoneme were enclosed by a thick layer of granular material. Mitochondria of midpiece lie alongside the proximal centriole which forms a very short neck region and possess tubular cristae internally and concentric layers of cristae superficially. During this course a fibrous sheath surrounds the axoneme of mid and principal piece. At the end dissolution of longitudinal manchette takes place. The mitochondria then rearrange themselves around the proximal and distal centrioles to form a neck region. Later, the fibrous sheath surrounds the proximal portion of the flagella. This part along with sperm head of D. zarudnyi provides a classical model that could be used in future for evolutionary and phylogenetic purposes of class reptilia.     

The spermatogenesis and the sperm structure of Terebrantia (Thysanoptera, Insecta)

Spermatogenesis and the sperm structure of the terebrantian Aeolothrips intermedius Bagnall are described. Spermatogenesis consists of two mitotic divisions; the second is characterized by the loss of half of the spermatids, which have pyknotic nuclei. Early spermatids have two centrioles, but when spermiogenesis starts, a third centriole is produced. The three basal bodies give rise to three flagella; later these fuse into a single flagellum which contains three 9 + 0 axonemes. The basal bodies are surrounded by a large amount of centriole adjunct material. During spermiogenesis this material contributes to the shifting of the three axonemes towards the anterior sperm region parallel to the elongating nucleus, and it is transformed into a dense cylinder. In the mature spermatids the three axonemes amalgamate to create a bundle of 27 doublet microtubules. Near the end of spermiogenesis the dense cylinder of the centriole adjunct lies parallel to the nucleus and the axonemes. It ends where the mitochondrion appears at half-sperm length. We confirm that Terebrantia testes have a single sperm cyst; their sperm are characterized by a cylindrical nucleus, three axonemes fused into one, a small mitochondrion and a short cylindrical centriole adjunct which corresponds to the dense body described in a previous work. The acrosome is lacking. At the midpoint of the anterior half of the sperm the outline of the cross-section is bilobed, with the nucleus contained in a pocket evagination of the plasma membrane. These characters are discussed in light of a comparison between Tubulifera and Terebrantia.     

瘤背石磺的精子结构

用光学显微镜和透射电子显微镜技术研究了瘤背石磺精子的结构特点,分析了其生理生态适应性以及在肺螺亚纲系统演化中的意义。瘤背石磺的精子由头部、中段和末段组成。头部由奶嘴形的顶体和长圆筒状的细胞核构成。顶体包括顶体囊和顶体构架体两部分;两者的内含物都分布均匀,电子密度稍低于细胞核;顶体基部平整,与核前端之间有一空隙,内含物电子密度极低。细胞核由电子密度高的均匀颗粒物质组成,并出现核泡;核的后端有一"杯形"的凹陷,称为核后窝。中段结构复杂,主要包括一对位于核后窝内的中心粒、轴丝、质膜、线粒体及由线粒体衍生的糖原质螺旋体、基质层和类晶体层等。末段由"9 2"结构的轴丝及外包的质膜组成,无糖原质螺旋体和其它线粒体衍生物。比较瘤背石磺精子与肺螺亚纲其它物种的精子结构,我们认为该物种的精子属于"进化型",是一类在进化地位中比基眼目高等的动物。     

Spermiogenesis in an iceryine coccid,Steatococcus tuberculatus morrison

The spermatozoon of Steatococcus is a motile filament containing a core of two chromosomes arranged in tandem and surrounded by more than 80 microtubules in 2 1/2 concentric rings. Two sperm develop from each binucleate spermatid in the form of long papillae. From the zone corresponding to the pole of the previous division microtubules appear and lengthen, assembly apparently occurring at their proximal undifferentiated ends. As they extend, they presumably push out the cytoplasmic papilla and co-extend a nuclear papilla through bridges with the nuclear envelope. Chromatin, attached to the envelope, is thus carried into the papilla, the shorter chromosome in the lead. 100 Å chromatin filaments are reduced to 20 Å and aligned as they enter the papilla. The filaments transform into 100 Å tubular fibrils, presumably by supercoiling. These then pack hexagonally, aggregate further into packed axial filaments, and finally condense into a nearly solid core in the mature sperm. Completed papillae (sperm) detach from the spermatid leaving behind nuclei devoid of chromatin. Following cycles of spiralization and despiralization, the sperm are bundled into hexagonal packs of 32 in register by cyst wall cells. The latter form primary and secondary sheaths and lay down a matrix within the bundle. As originally reported by Hughes Schrader (1946), no evidence of centriole, acrosome, mitochondrial derivative or structure suggesting flagellar axoneme is found in either the developing papilla or the mature sperm. The microtubules determine the axis of the developing sperm; polarity is set by the direction of sperm motion and is homologous with most flagellate sperm in that the nuclear material is anterior and the microtubule initiating center is posterior. All of the functions attributed to microtubules are manifest in differentiation of this sperm: extension, support, translocation and motility.This paper is affectionately dedicated to Professor Sally Hughes-Schrader on the occasion of her seventy-fifth birthday, with warm appreciation of her friendship, her exemplary science, her keen criticism, her contagious enthusiasm, and for leading me to Steatococcus.     

INFLUENCE OF THE MULTILAYERED STRUCTURE ON THE MORPHOGENESIS OF MARCHANTIA SPERMATIDS

The multilayered structure (MLS) in a spermatid of Marchantia is the morphogenetic blueprint of the headpiece in a mature sperm. As the nucleus begins elongation, a curved, tapered nuclear projection follows the path of microtubules extending from the MLS and becomes inserted into an indented zone at the rear of the asymmetric organelle. The indented zone defines the most forward penetration of the nucleus into the sperm headpiece. Partial disorganization of MLS lower strata nearest the nuclear projection facilitates overlapping of the nucleus with the rearward part of the anterior mitochondrion. At the front of the nascent headpiece, the mitochondrion is stabilized against microtubules following total disorganization of intervening MLS strata. Penetration of the nuclear projection along the MLS and directed disorganization of MLS lower strata control ultimate disposition of headpiece components. The headpiece is isolated and molded into final shape by undercutting and constriction of the cell membrane.     

Chromatin organization during spermiogenesis in Octopus vulgaris. I: Morphological structures

In the process of the chromatin remodeling that occurs during spermiogenesis in some animal species, it is possible to distinguish between two separate aspects: the chromatin condensation pattern itself (granular, fibrillar, or lamellar), and the architecture of this pattern, that is to say, its arrangement within the nucleus. In the cephalopod Octopus vulgaris these two aspects are clearly differentiated. The condensation pattern develops from 25 nm fibers to fibers with a tubular aspect and with a progressively increasing diameter (40-60 nm and then to 80 nm), to end finally in the form of very thin fibers (3-5 nm) product of the coalescence and dissolution of the major fibers. The main directive force that governs this process lies in the global change that occurs in the proteins that interact with all (or the major part) of the genomic DNA. The condensation pattern by itself in this species does not present a fixed order: most of the fibers appear without any predominant spatial direction in the spermiogenic nuclei. However, as the nuclei elongate, the chromatin fibers arrange in parallel following the elongation axis. This parallel disposition of the chromatin fibers appears to be mediated by two specific areas, each of which we call a "polar nuclear matrix" (PNM). These matrices differentiate in the basal and apical nuclear poles adjacent to the centriolar implantation fosse and the acrosome, respectively. The areas that constitute the PNM have the following characteristics: (a) they are the only areas where DNA is found anchored to the nuclear membrane; (b) they are the zones from which the chromatin condensation pattern (fibers/tubules) begins; and (c) they are most probably the points through which the mechanical forces originating from nuclear elongation are transmitted to chromatin, causing the chromatin fibers/tubules to adopt an almost perfectly parallel disposition. Finally, we discuss the importance of the architecture of the chromatin condensation pattern, as it is one of the determining factors of the spatial organization of the mature sperm genome and chromosome positioning.