Neck region of gerbil spermatozoa

In the course of the reorganization and degeneration of the proximal centriole in the mature acentriolate spermatozoon of the Mongolian gerbil, both the proximal and distal centrioles appear in the early cap phase of spermatid development. During the acrosome phase, both distal and proximal centrioles become highly active in the formation of a segmented column. The proximal centriole becomes actively involved in the formation of the capitulum, while the distal centriole forms the axonemal complex and dense fibers. During the maturation phase of spermatid development, the “pinwheel” arrangement of the proximal centriole becomes an “S”-shaped structure, turned 90° on its vertical axis. The few “doublet” microtubules that can be detected later in that stage completely disappear during spermiation. The distal centriolar area develops a single central pair of microtubules and membranous elements. Another prominent feature in the neck region of the gerbil spermatozoa is the presence of two dense rudimentary columns in association with the mitochondria. Although their density is similar to that of the other columns, these two columns have no connection with the dense fibers; in fact, they are closely associated with the mitochondria.     

The fine structure of the spermatozoon of Pennaria tiarella (coelenterata)

Spermatozoa of the hydroid Pennaria tiarella were examined with the electron microscope. The anterior region is characterized by the presence of 30–40 membrane-bounded vesicles which lie anterior to the nucleus. These vesicles are apparently derived from the Golgi apparatus. The nucleus is conical in shape with a protrusion at the anterior end. Posteriorly it is indented by four radially arranged mitochondria. Lying within the fossa formed by the mitochondria are proximal and distal (filament forming) centrioles. The distal centriole is characterized by nine centriole satellite projections which emanate from its matrix. The tubules of the distal centriole are continuous with the alpha filaments of the tail. The tails are typical 9 + 2 flagella with 9 peripheral doublet (or alpha) filaments surrounding two central (or beta) filaments.     

The behaviour of centrioles and the formation of the flagellum in rooster and drake spermatids

Summary Developmental changes in the formation of the centrioles and flagellum during spermiogenesis in the rooster and drake were studied.Changes in the length and thickness of the wall of the centrioles were observed from an early stage of spermatid development. Before the proximal centriole is attached to the nucleus microtubules were observed near the centrioles joined to them. At this stage of spermatid development changes on the nuclear membrane were observed at a place where the proximal centriole is attached to the nucleus. At the later stage of spermatid differentiation three to five dense extensions in the space of the nuclear invagination and dense bodies or granules near the distal centriole were present. The anterior part of the newly formed flagellum is covered by a cytoplasmic membrane displaying extension which is approximately 1.3 m long. Slight differences between the two species were observed.     

STUDIES OF SPERMATOGENESIS IN THE HEPATICAE V. BLEPHAROPLAST DEVELOPMENT IN MARCHANTIA POLYMORPHA

Coaxial centrioles and a microtubule organizing center (MTOC) constitute each centrosome in spermatid mother cells of Marchantia polymorpha. During cell division the centrosome separates at its midregion and the two centrioles undergo a planar rotation that brings them to lie somewhat staggered and nearly parallel with their proximal ends embedded in osmiophilic granular material similar in appearance to that of the MTOC. Microtubules of the multilayered structure (MLS) arise in this material below the posterior centriole and parallel to its long axis. The rotation of centrioles and the initiation of S₁ tubules below the posterior centriole determine polarity of the incipient blepharoplast. Lower MLS strata are formed under the anterior centriole by the compaction of granular, osmiophilic matrix. Formation and growth of S₂ vertical lamellae occur at the left front edge of the MLS in association with MTOC-like matrix localized near the cell membrane. The MLS enlarges to about 0.4 μm wide by 0.6 μm long and is ovoid in outline except for a short distal projection underlying the posterior centriole. Subsequently the lamellae are transformed into homogenous, osmiophilic matrix that contributes directly to the expansion of all MLS strata including microtubules. The stratum of lamellae is interpreted as a planar MTOC subject to morphogenetic control. Each of the four strata grows proximally while the tapering distal projection lengthens beneath the posterior basal body. Dense matrix above the MLS, apparently elaborated by the S₂ layer, is organized into cartwheel and triplet components of the basal bodies’ proximal extensions. Organization of triplet tubules proceeds from proximal to distal toward preexisting triplets. Osmiophilic matrix contributes to the formation of microtubule keels and osmiophilic crests and may serve as a cementing material that stabilizes the spatial relationships of blepharoplast components. After full expansion of the MLS’ lower strata, the S₂ layer is reorganized into lamellae. Flagellar growth in Marchantia is postulated to involve a process whereby subunits or their precursors are elaborated by the MLS, translocated to the distal end of the flagellum and incorporated into the axonemal tubules. When MLS microtubules elongate to form a long, narrow band, the distal half of the S₂ layer is again in the osmiophilic matrix state.     

A ciliopathy complex builds distal appendages to initiate ciliogenesis

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and superresolved imaging, we identify a module that we term DISCO (distal centriole complex). The DISCO components CEP90, MNR, and OFD1 underlie human ciliopathies. This complex localizes to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR do not generate cilia, fail to assemble distal appendages, and do not transduce Hedgehog signals. Disrupting the satellite pools does not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruits the most proximal known distal appendage component, CEP83, to root distal appendage formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricts centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies.     

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis

Cilia are found on most human cells and exist as motile cilia or non-motile primary cilia. Primary cilia play sensory roles in transducing various extracellular signals, and defective ciliary functions are involved in a wide range of human diseases. Centrosomes are the principal microtubule-organizing centers of animal cells and contain two centrioles. We observed that DNA damage causes centriole splitting in non-transformed human cells, with isolated centrioles carrying the mother centriole markers CEP170 and ninein but not kizuna or cenexin. Loss of centriole cohesion through siRNA depletion of C-NAP1 or rootletin increased radiation-induced centriole splitting, with C-NAP1-depleted isolated centrioles losing mother markers. As the mother centriole forms the basal body in primary cilia, we tested whether centriole splitting affected ciliogenesis. While irradiated cells formed apparently normal primary cilia, most cilia arose from centriolar clusters, not from isolated centrioles. Furthermore, C-NAP1 or rootletin knockdown reduced primary cilium formation. Therefore, the centriole cohesion apparatus at the proximal end of centrioles may provide a target that can affect primary cilium formation as part of the DNA damage response.     

Klp10A modulates the localization of centriole-associated proteins during Drosophila male gametogenesis

Mutations in Klp10A, a microtubule-depolymerising Kinesin-13, lead to overly long centrioles in Drosophila male germ cells. We demonstrated that the loss of Klp10A modifies the distribution of typical proteins involved in centriole assembly and function. In the absence of Klp10A the distribution of Drosophila pericentrin-like protein (Dplp), Sas-4 and Sak/Plk4 that are restricted in control testes to the proximal end of the centriole increase along the centriole length. Remarkably, the cartwheel is lacking or it appears abnormal in mutant centrioles, suggesting that this structure may spatially delimit protein localization. Moreover, the parent centrioles that in control cells have the same dimensions grow at different rates in mutant testes with the mother centrioles longer than the daughters. Daughter centrioles have often an ectopic position with respect to the proximal end of the mothers and failed to recruit Dplp.     

Achromatic Figure Formation by Multiple Centrioles of Barbulanympha

SUMMARY. The role of centrioles in achromatic figure production is considered when the number present varies from 1–8. Each centriole after it becomes elongate produces astral rays from its distal end. Some of these rays remain free; some, by joining centromeres, become chromosomal fibers; and some, by joining and growing along those produced by one or more other centrioles, produce the central spindle portion of the achromatic figures. Thus, one centriole may function cooperatively with one to several others in the production of central spindles. But at least two centrioles must be present, and in the proper spacial relation to each other, to form a central spindle; one by itself can form only free astral rays, no central spindle or chromosomal fibers.
The flagellated areas (to which the centrioles are anchored anteriorly) play an important role in determining the position of the distal ends of the centrioles with respect to one another, and the position of these ends, in turn, in a large measure, determines the types of achromatic figures produced, particularly the number of central spindles.     

Centrioles in the cell cycle. I. Epithelial cells

A study was made of the structure of the centrosome in the cell cycle in a nonsynchronous culture of pig kidney embryo (PE) cells. In the spindle pole of the metaphase cell there are two mutually perpendicular centrioles (mother and daughter) which differ in their ultrastructure. An electron-dense halo, which surrounds only the mother centriole and is the site where spindle microtubules converge, disappears at the end of telophase. In metaphase and anaphase, the mother centriole is situated perpendicular to the spindle axis. At the beginning of the G1 period, pericentriolar satellites are formed on the mother centriole with microtubules attached to them; the two centrioles diverge. The structures of the two centrioles differ throughout interphase; the mother centriole has appendages, the daughter does not. Replication of the centrioles occurs approximately in the middle of the S period. The structure of the procentrioles differs sharply from that of the mature centriole. Elongation of procentrioles is completed in prometaphase, and their structure undergoes a number of successive changes. In the G2 period, pericentriolar satellites disappear and some time later a fibrillar halo is formed on both mother centrioles, i.e., spindle poles begin to form. In the cells that have left the mitotic cycle (G0 period), replication of centrioles does not take place; in many cells, a cilium is formed on the mother centriole. In a small number of cells a cilium is formed in the S and G2 periods, but unlike the cilium in the G0 period it does not reach the surface of the cell. In all cases, it locates on the centriole with appendages. At the beginning of the G1 period, during the G2 period, and in nonciliated cells in the G0 period, one of the centrioles is situated perpendicular to the substrate. On the whole, it takes a mature centriole a cycle and a half to form in PE cells.     

OBSERVATIONS ON THE FINE STRUCTURE OF THE DEVELOPING SPERMATID IN THE DOMESTIC CHICKEN

The kinetic apparatus, the acrosome and associated structures, and the manchette of the spermatid of the domestic chicken have been studied with the electron microscope. The basic structural features of the two centrioles do not change during spermiogenesis, but there is a change in orientation and length. The proximal centriole is situated in a groove at the edge of the nucleus and oriented normal to the long axis of the nucleus and at right angles to the elongate distal centriole. The tail filaments appear to originate from the distal centriole. The plasma membrane is invaginated along the tail filaments. A dense structure which appears at the deep reflection of the plasma membrane is identified as the ring. The fine structure of the ring has no resemblance to that of a centriole and there is no evidence that it is derived from or related to the centrioles. The tail of the spermatid contains nine peripheral pairs and one central pair of tubular filaments. The two members of each pair of peripheral filaments differ in density and in shape: one is dense and circular, and the other is light and semilunar in cross-section. The dense filaments have processes. A manchette consisting of fine tubules appears in the cytoplasm of the older spermatid along the nucleus, neck region, and proximal segment of the tail. The acrosome is spherical in young spermatids and becomes crescentic and, finally, U-shaped as spermiogenesis proceeds. A dense granule is observed in the cytoplasm between acrosome and nucleus. This granule later becomes a dense rod which is interpreted as the perforatorium.     

Fine structure of an elongated dorso-ventrally compressed echinoderm (holothuroidea) spermatozoon

The spermatozoon of Cucumaria pseudocurata is unique among those of the echinoderms in that it is tabloid in shape, i.e., elongated and dorsoventrally compressed. The sperm consists of a dorsal surface which contains an extensive striated rootlet-like structure located within a dorsal groove and a ventral surface which contains a medially situated acrosome. A single mitochondrion lies at the base of the nucleus. The flagellum is unusual in that a 9 + 3 tubular arrangement is observed in the mid-tail region. The acrosome consists of an acrosomal granule bounded by a limiting membrane and a surrounding periacrosomal layer. The granule is irregular in shape with the anterior-posterior surfaces flaring out, forming pockets in the periacrosomal material. The ventral granule surface bulges forming a close association with the plasma membrane. The dorsal surface is indented. Ventral to the depression (within the granule) is a small area containing a particulate-fibrous material. To the inside of the granule limiting membrane there is a second membrane-like structure (incomplete) which extends from the anterior-posterior surfaces around the dorsal face of the granule. Dorso-medial to the granule the periacrosomal layer contains a particulate-fibrous region lodged within the granule depression. This material is presumably the precursor of the acrosomal filament. Prominent cytoplasmic folds extend off from the basal flagellar region. The proximal and distal centrioles are situated perpendicular to one another within the mitochondrion. Centriolar satellite materials are associated with both centrioles. Toward the base of the tail the satellite of the distal centriole consists of nine radiating arms extending at an angle of 45° to the axis of the centriole. Each arm terminates in a dense thickening. The striated rootlet extends anteriorly from the distal centriole to just below the level of the acrosome.     

Ultrastructure of spermiogenesis in the gastropod Calliotropis glyptus Watson (Prosobranchia: Trochidae)with special reference to the embedded acrosome

Testicular spermatozoa and sperm development in the archaeogastropod Calliotropis glyptus Watson (Trochoidae: Trochidae) are examined using transmission electron microscopy and formalin-fixed tissues. During spermiogenesis, the acrosome, formed evidently through fusion of Golgi-derived proacrosomal vesicles, becomes deeply embedded in the condensing spermatid nucleus. Two centrioles (proximal and distal), both showing triplet microtubular substructure, are present in spermatids—the distal centriole giving rise to the sperm tail and its associated rootlet. During formation of the basal invagination in the spermatid nucleus, centrioles, and rootlet move towards the nucleus and come to lie totally within the basal invagination. Mitochondria are initially positioned near the base of the nucleus but subsequently become laterally displaced. Morphology of the mature spermatozoon is modified from that of the classic primitive or ect-aquasperm type by having 1) the acrosome embedded in the nucleus (the only known example within the Mollusca), 2) a deep basai invagination in the nucleus containing proximal and distal centrioles and an enveloping matrix (derived from the rootlet), 3) laterally displaced periaxonemal mitochondria, and 4) a tail extending from the basal invagination of the nucleus. Implantation of the acrosomal complex and centrioles within imaginations of the nucleus and lateral displacement of mitochondria effectively minimize the length of the sperm head and midpiece. Such modifications may be associated with motility demands, but this remains to be established. The unusual features of C. glyptus spermatozoa, though easily derivable from ‘typical’ trochoid sperm architecture, may prove useful in delineating the genus Calliotropis or tracing its relationship to other genera within the trochid subfamily Margaritinae.     

Spermiogenesis in Polyplacophora,with special reference to acrosome formation (Mollusca)

Summary The present study examines spermiogenesis, and in particular the formation of the acrosome, in ten species of chitons belonging to four families. This study emphasizes the formation of the acrosome but brings to light several other structures that have received little or no mention in previous studies. The process of spermiogenesis is essentially similar in each species, although Chaetopleura exhibits some significant differences. In early spermiogenesis the Golgi body secretes numerous small pro-acrosomal vesicles that gradually migrate into the apical cytoplasm. The chromatin condenses from granules into fibres which become twisted within the nucleus. A small bundle of chromatin fibres projects from the main nuclear mass into the anterior filament; this coincides with the appearance of a developing manchette of microtubules around the nucleus that originates from the two centrioles. Radiating from the distal centriole is the centriolar satellite complex, which is attached to the plasma membrane by the annulus. The distal centriole produces the flagellum posteriorly and it exits eccentrically through a ring of folded membrane that houses the annulus. Extending from the annulus on one side of the flagellum, in all but one species, is a dense fibrous body that has not been previously reported. The proximal centriole lies perpendicular to the end of the distal centriole and is attached to it by fibro-granular material. Pro-acrosomal vesicles migrate anteriorly through the cytoplasm and move into the anterior filament to one side of the expanding nucleus. Eventually these vesicles migrate all the way to the tip of the sperm, where they fuse to form one of two granules in the acrosome. In mature sperm the nucleus is bullet-shaped with a long anterior filament and contains dense chromatin with occasional lacunae. The mitochondria vary in both number and position in the mature sperm of different species. Both centrioles are housed eccentrically in a posterior indentation of the nucleus, where the membranes are modified. The elongate flagellum tapers to a long filamentous end-piece that roughly corresponds to the anterior filament and may be important in sperm locomotion for hydrodynamic reasons. An acrosome is present in all ten species and stained positively for acid phosphatase in three species that were tested.     

The centriole cycle in the amoebae of the myxomycetePhysarum polycephalum

Summary In the amoebae of the myxomycetePhysarum polycephalum, procentrioles are formed on the anterior and posterior centrioles in early prophase. Although the relative position of the parental and procentrioles is fixed, all relative positions of the daughter and parental centrioles were observed. During the different stages of mitosis daughter centrioles elongate and acquire anterior satellites, one of the characteristic features of the anterior centrioles. All other anterior morphological characteristics appear only in telophase and early reconstruction stages. In contrast to the parental posterior centrioles, which do not change morphologically during the successive mitotic stages, the parental anterior centrioles lose their morphological characteristics in late prophase and early prometaphase and then acquire the morphological features characteristic of the posterior centrioles. Thus, the following maturation scheme is suggested: a procentriole becomes an anterior centriole during the first mitosis and a posterior centriole during the second mitosis. Since posterior features are maintained during mitosis, the posterior centriole corresponds to the final state of centriole maturation.     

OBSERVATIONS ON THE CENTRIOLES OF THE SEA URCHIN SPERMATOZOON

The spermatozoon of Lytechinus variegatus has two parallel centrioles. The basal body of the flagellum consists of the proximal centriole (a short cylinder of nine tubule-triplets) and its distal extension of nine tubule-doublets. The distal centriole lies near the distal end of the basal body, between the nucleus and the mitochondrion. The observations suggest that both the proximal and the distal centrioles are polarized structures, their tubule-triplets pitched in the same direction and their distal ends associated with the flagellar axoneme and with the mitochondrion, respectively. The distal centriole in different spermatozoa occupies different positions around the basal body-flagellum complex.     

CENTRIOLE REPLICATION : A Study of Spermatogenesis in the Snail Viviparus

This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut from pieces of testis fixed in OsO₄ and embedded in the polyester resin Vestopal W. Two kinds of spermatocytes are present. These give rise to typical uniflagellate sperm carrying the haploid number of 9 chromosomes, and atypical multiflagellate sperm with only one chromosome. Two centrioles are present in the youngest typical spermatocyte. Each is a hollow cylinder about 160 mµ in diameter and 330 mµ long. The wall consists of 9 sets of triplet fibers arranged in a characteristic pattern. Sometime before pachytene an immature centriole, or procentriole as it will be called, appears next to each of the mature centrioles. The procentriole resembles a mature centriole in most respects except length: it is more annular than tubular. The daughter procentriole lies with its axis perpendicular to that of its parent. It presumably grows to full size during the late prophase, although the maturation stages have not been observed with the electron microscope. It is suggested that centrioles possess a constant polarization. The distal end forms the flagellum or other centriole products, while the proximal end represents the procentriole and is concerned with replication. The four centrioles of prophase (two parents and two daughters) are distributed by the two meiotic divisions to the four typical spermatids, in which they function as the basal bodies of the flagella. Atypical spermatocytes at first contain two normal centrioles. Each of these becomes surrounded by a cluster of procentrioles, which progressively elongate during the late prophase. After two aberrant meiotic divisions the centriole clusters give rise to the basal bodies of the multiflagellate sperm. These facts are discussed in the light of the theory, first proposed by Pollister, that the supernumerary centrioles in the atypical cells are derived from the centromeres of degenerating chromosomes.     

Plk4-induced centriole biogenesis in human cells

We show that overexpression of Polo-like kinase 4 (Plk4) in human cells induces centrosome amplification through the simultaneous generation of multiple procentrioles adjoining each parental centriole. This provided an opportunity for dissecting centriole assembly and characterizing assembly intermediates. Critical components were identified and ordered into an assembly pathway through siRNA and localized through immunoelectron microscopy. Plk4, hSas-6, CPAP, Cep135, gamma-tubulin, and CP110 were required at different stages of procentriole formation and in association with different centriolar structures. Remarkably, hSas-6 associated only transiently with nascent procentrioles, whereas Cep135 and CPAP formed a core structure within the proximal lumen of both parental and nascent centrioles. Finally, CP110 was recruited early and then associated with the growing distal tips, indicating that centrioles elongate through insertion of alpha-/beta-tubulin underneath a CP110 cap. Collectively, these data afford a comprehensive view of the assembly pathway underlying centriole biogenesis in human cells.     

Stress-induced localization of HSPA6 (HSP70B′) and HSPA1A (HSP70-1) proteins to centrioles in human neuronal cells

The localization of yellow fluorescent protein (YFP)-tagged HSP70 proteins was employed to identify stress-sensitive sites in human neurons following temperature elevation. Stable lines of human SH-SY5Y neuronal cells were established that expressed YFP-tagged protein products of the human inducible HSP70 genes HSPA6 (HSP70B′) and HSPA1A (HSP70-1). Following a brief period of thermal stress, YFP-tagged HSPA6 and HSPA1A rapidly appeared at centrioles in the cytoplasm of human neuronal cells, with HSPA6 demonstrating a more prolonged signal compared to HSPA1A. Each centriole is composed of a distal end and a proximal end, the latter linking the centriole doublet. The YFP-tagged HSP70 proteins targeted the proximal end of centrioles (identified by γ-tubulin marker) rather than the distal end (centrin marker). Centrioles play key roles in cellular polarity and migration during neuronal differentiation. The proximal end of the centriole, which is involved in centriole stabilization, may be stress-sensitive in post-mitotic, differentiating human neurons.