Thesis (M.S. in genetics and cell biology)--Washington State University, December 2008. / Title from PDF title page (viewed on Dec. 31, 2008). "School of Molecular Biosciences." Includes bibliographical references.

Sperm structure in five species of cicadine cicadas (Albanycada albigera, Azanicada zuluensis, Platypleura capensis, P. hirtipennis and Pycna semiclara) and five species of cicadettine cicadas (Melampsalta leucoptera, Quintilia walkeri, Stagira simplex, Xosopsaltria thunbergi and Monomatapa matoposa) was investigated by light and electron microscopy. In addition, spermiogenesis in cicadas was described; the information was derived from two cicadettines (Diceroprocta biconica and M. matoposa) and three cicadines (Kongota punctigera, P. capensis and P. semiclara). Mature spermatozoa of all species investigated are elongate and filiform, consisting of three distinct regions: the head (acrosome and nucleus), mid-piece and tail. All species produce more than one discrete length of nucleated, motile sperm, a form of sperm polymorphism termed polymegaly. Polymegaly is expressed in three ways; sperm have uni-, bi- or trimodal nucleus and tail lengths. Besides the differences in length, there are also notable differences in the size of nuclei. The anterior parts of sperm heads are embedded in an elongate homogenous matrix forming spermatodesmata. The conical acrosome is deeply invaginated posteriorly, and sits on top of the nucleus. The acrosomal contents are differentiated internally with a tubular substructure and a subacrosomal space. The anterior of the nucleus intrudes into the posterior section of the subacrosomal space. Anteriorly the acrosome is laterally flattened; posteriorly it extends as two tubular processes on either side of the nucleus that gradually decrease in diameter. The homogenously electron-dense nucleus is pointed anteriorly and is generally cylindrical, although posteriorly there is a lateral invagination that extends part-way along the nucleus. This invagination houses fine granular material of the putative centriolar adjunct which does not form in close proximity to the centriole and hence may not be a true centriolar adjunct. The lamellate disposition of the centriolar adjunct material within the sperm-midpiece of cicadettine cicadas is distinct, and separates these cicadas from their cicadine counterparts in which the centriolar adjunct material is non-lamellate. Vesicle-like elements that are associated with both the posterior nucleus and the centriolar adjunct are also found within the invagination. Immediately posterior of and adjoining the centriolar adjunct is a pair of mitochondrial derivatives that are elongated and extend for almost the entire length of the tail. Except for size the architecture of short and long spermatozoa is generally similar in all species. The absence of accessory bodies in cicada sperm suggests that within the Cicadomorpha, the families Cicadidae and Cercopidae are closely related. Only long nuclei were observed in the fertilized eggs of A. zuluensis indicating that sperm with long nuclei might be favoured for fertilization. Spermiogenesis involves: (a) development of the acrosome from a proacrosomal granule; (b) development of the nucleus, characterized by elongation and streamlining with a simultaneous condensation of chromatin; (c) development of the axoneme from the centriole; (d) amalgamation of individual small mitochondria to form elongated mitochondrial derivatives in which cristae are arranged into regularly spaced lamellae; and (f) elimination of cytoplasm. The presence of a manchette, a transient microtubular organelle, which surrounds the acrosome, nucleus and mitochondrial derivatives, is a characteristic feature of spermiogenesis. The gross morphology of the reproductive tract in both male and female cicadas exhibits an organization similar to that in most oviparous insects. The non-functional spermatheca is the only exceptional feature in the female reproductive tract. Its role has been taken over by the common oviduct which, subsequently, has become modified into a swollen, differentiated structure with a dual role of receiving oocytes from the paired ovaries and storage of spermatozoa. Testis mass varies between cicada species; this variation might be linked to the intensity of sperm competition which has been found to be positively correlated with relative investment in spermatogenesis. Based on the preliminary findings of this study, K. punctigera, with its larger testis relative to body size, would be the ideal candidate to show the greatest levels of sperm competition. Accessory glands in both male and female A. zuluensis, D. biconica, P. hirtipennis and O. quadraticollis are very long; this character might be of phylogenetic significance. Despite being notoriously refractory spermiocladistics is potentially valuable in systematic and phylogenic studies of cicadas, especially at the subfamily level.

Specialized in form and function, the sperm cell is a unique microsystem unto itself where in the cytoskeletal processes and structures of the somatic cell often find new purpose and characteristics within the sperm. Unlike other cells in the human body, this unique cell polarizes and transforms itself from a line of germ cells to evolve into a functional, hydrodynamic haploid spermatozoon. The success of fertilization is dependent on this haploid cell and its specially designed vesicular structure, the acrosome, which provides the leading edge of oocyte penetration. To date, there is little insight into the mechanics of how acrosomic vesicles are successfully targeted and transported to the nuclear envelope and tether to its surface. Our laboratory has identified a novel 15 kDa sperm specific histone variant, SubH2Bv, which possesses a distinct and functional nuclear localization signal (NLS) that associates with the acrosomic vesicle. This study provides evidence that SubH2Bv’s bipartite NLS (an NLS with two basic domains linked together by 10-12 amino acid residues) is responsible for directing acrosomic vesicles to the nuclear envelope using the somatic import receptor, karyopherin alpha (Kap α). Based on bipartite NLS-receptor conventions, where karyopherin alpha is known to specifically associate with this NLS-type, SubH2Bv would be the karyophilic cargo and karyopherin alpha would act as part of the underlying transport mechanism. Western blot analysis and immunohistochemistry characterized Kap α as a membrane-associated sperm protein that is co-localized with SubH2Bv around the proacrosomic granules and the acrosomic vesicle during spermiogenesis. Their co-expression and co-localization, as demonstrated by immunolabelling, suggested a potential binding relationship that was confirmed by a His-tag-recombinant SubH2Bv-pull-down assay. The co-developmental acrosomic expression of Kap α and SubH2Bv in haploid cells, combined with the pull-down evidence of their binding affinity, provides a compelling argument that these two proteins work in concert to traffic the acrosomic vesicles to the nucleus. The exclusion of these two otherwise nuclear proteins from the nucleus, and their co-localization to the subacrosomal region in elongating spermatids, also implies a contingent role for SubH2Bv and Kap α in acrosomal docking, that may involve the classical bipartite/Kap α nuclear import pathway. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2008-09-02 16:11:17.429

Spermiogenesis and ultrastructure of the spermatozoon of two bothriocephalidean cestodes, Oncodiscus sauridae and Senga sp., have been studied using transmission electron microscopy. The presence of a classical pattern for spermatological characters (spermiogenesis of type I with dense-material in early stages and sperm of type II with a characteristic ring of cortical microtubules in the anterior part) in Bothriocephalidea is discussed.

No description available.

Les gènes du chromosomeX sont majoritairement inactivés au cours de la méiose mâle. Chez la souris, seulement 6% d’entre eux sont réactivés au cours des stades post-méiotiques. Parmi eux le gène Uba1X codant pour l’enzyme activatrice de l'ubiquitine, UBA1 qui produit trois transcrits dont deux sont ubiquitaires mais le troisième prédomine dans les cellules post¬-méiotiques : les spermatides. Nos travaux montrent que les 5’UTR, seul différence entre ces trois transcrits, déterminent la localisation et la dose relative des isoformes nucléaire et cytoplasmique de la protéine UBA1. Nous avons mis en évidence chez la souris que le transcrit spermatide-spécifique code pour l'isoforme nucléaire, exprimée fortement dans les spermatides suggérant un rôle de la protéine UBA1 dans la dégradation des histones lors du remodelage chromatinien. Nous avons détecté deux mutations dans la région spermatide-spécifique du gène : une délétion de 13pb et une transition G>A, chacune portée par un patient infertile, et non retrouvée dans notre population témoin. Les analyses ont montré que la délétion de 13pb induit un épissage anormal du transcrit spermatide-spécifique et que la transition G>A pouvait réduire le taux d’expression du transcrit spermatide-spécifique. Ces mutations pourraient induire l'infertilité des deux patients. En parallèle nous avons pu démontrer que les mutations dans le gène codant pour la protamine PRM1 décrites dans la littérature c.102G>T et c.-107G>C ne sont pas liées à l'infertilité masculine et que le variant est un polymorphisme fréquemment retrouvé dans la population congolaise. / The majority of genes on the X chromosome are repressed during meiosis and only 6% of them are expressed in post meiotic germ cells. One of these genes is Ubal, encoding the ubiquitin-activating enzyme UBA1. Ubal produces three different transcripts, two of which are ubiquitously expressed while the third is predominant in the post meiotic germ cells: the spermatids. Our study shows that the 5’UTR, which is the only difference between these transcripts, determines the localization and the relative dose of the nuclear and cytoplasmic isoform of the UBA1 protein. The spermatid-specific transcript encodes for the nuclear isoform in the spermatids in the mouse suggesting that the UBA1 protein is implicated in chromatin remodeling during spermiogenesis. We have detected two mutations in the spermatid-specific region of the UBA1 gene in two infertile men: a deletion of 13bp and a G>A transition, neither of which was found in our cohort of fertile men. The deletion of 13bp diminishes the correct splicing of the spermatid-specific transcript and that the G>A transition may reduce expression of the spermatid-specific transcript. These results show that the UBA1 gene is involved in spermiogenesis, and reactivated in spermatids by its spermatid-specific transcript and that the mutations identified may induce infertility by reducing UBA1 levels in spermatids. We have also demonstrated that two variants described in the protamine codant gene PRM1C.102G>T and c.-107G>C are clearly not associated with male infertility and that the c.-107G>C is polymorphism frequently found in the congolese population.

Orientadores: Irani Quagio Grassiotto, Luiz Roberto Malabarba / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T19:44:29Z (GMT). No. of bitstreams: 1 Baicere-Silva_ClariannaMartins_D.pdf: 11314225 bytes, checksum: beda03a1ee3e0b2d960e66de41e95061 (MD5) Previous issue date: 2013 / Resumo: A família Characidae apresenta 16 subfamílias e noventa e dois gêneros não assinalados a nenhuma subfamília e, portanto, considerados incertae sedis. Neste grupo são inúmeros os problemas taxonômicos/filogenéticos. A maior parte destes gêneros encontravam-se alocados na subfamília Tetragonopterinae, mas dada à falta de evidências de que Tetragonopterinae constitui um grupo monofilético, manteve-se na subfamília apenas o gênero Tetragonopterus. Os gêneros incertae sedis, em Characidae, constituem um grupo imenso e heterogêneo de peixes, predominantemente pequenos, abundantes nos rios e em outros habitats aquáticos da região neotropical. O conhecimento das relações de parentesco entre os Characidae e, consequentemente, entre os antigos Tetragonopterinae, tem por base principalmente características osteológicas, de anatomia de partes moles e, mais recentemente, dados de seqüência molecular. Sabe-se que as características reprodutivas podem conter importantes sinais filogenéticos. Portanto, procedeu-se uma análise filogenética a partir de dados reprodutivos como a estrutura testicular, o tipo de espermiogênese e a ultraestrutura dos espermatozoides de representantes de alguns dos gêneros incertae sedis em Characidae, anteriormente alocados em Tetragonopterinae e outros Characiformes utilizados como grupo externo. A topologia da árvore obtida, foi parcialmente congruente com as hipóteses existentes para o grupo, comprovando a utilidade deste tipo de dado no que se refere à elucidação dos padrões evolutivos em Characidae / Abstract: The family Characidae presents 16 subfamilies and ninety-two genera not reported to any subfamily and therefore considered incertae sedis. In this group there are numerous taxonomic / phylogenetic problems. Most of these genera were allocated in the subfamily Tetragonopterinae, but given the lack of evidence that Tetragonopterinae constitute a monophyletic group, remains the only genus Tetragonopterus in the subfamily. Genera incertae sedis in Characidae are a vast and heterogeneous group of fish, mostly small, abundant in rivers and other aquatic habitats of the Neotropics. Researches of relationships among the Characidae and consequently between the old Tetragonopterinae is based mainly in external morphollogical and osteological features and, more recently, molecular sequence data. It is known that reproductive characteristics may contain significant phylogenetic signals. So we proceeded from a phylogenetic analysis of reproductive data such as testicular structure, the type of spermiogenesis and sperm ultrastructure of representatives of some of the genera incertae sedis in Characidae, previously allocated to Tetragonopterinae and other Characiformes used as outgroup. The tree topology obtained was parcially congruent with the hypotheses exist for the group proving the usefulness of this type of data in relation to elucidate the evolutionary patterns in characid / Doutorado / Biologia Tecidual / Doutora em Biologia Celular e Estrutural

La spermatogenèse est le processus par lequel les cellules germinales sont transformées en spermatozoïdes par le déroulement de 3 phases: la phase mitotique et méiotiques et la spermiogénèse. Pendant la spermiogénèse d'importantes structures sont formées afin de générer un spermatozoïde fonctionnel : l’acrosome, la manchette et le flagelle. La manchette est une structure transitoire situé caudalement à l’acrosome, composée par un manteau de microtubules longeant le noyau du spermatide. La manchette est connue pour participer au remodelage du noyau afin de lui conférer une forme falciforme ainsi que pour son rôle dans le développement de l’acrosome et du flagelle. En effet, pendant la spermiogénèse toutes les molécules nécessaires pour la formation du flagelle et de l’acrosome doivent être transportées sur leur site d'assemblage. Les microtubules forment la manchette permettent le mouvement de protéines entre la région pré-acrosomique et la zone d’assemblage du flagelle. Cependant ce transport doit être finement régulé dans l’espace et dans le temps car la localisation aberrante et/ou manquante de certaines protéines peut causer des malformations de l’ acrosome, de la manchette et du flagelle. Un mécanisme qui peut expliquer la façon dont ce processus de transport peut être régulé est la génération de modification post-traductionnelles de la tubuline forment les microtubules car ces modifications peuvent réguler les interactions avec les moteurs moléculaires et les protéines associées aux microtubules. La polyglutamylation correspond à un attachement covalent de chaines de glutamates latérales sur la queue terminale de la tubuline. Cette modification est contrôlée par la coordination des enzymes glutamylase (TTLLs) et déglutamylase (CCPs). De récents études ont souligné l'importance potentielle de certaines de ces enzymes dans la formation et la maintenance du flagelle. Mon projet est centré sur l’étude des fonctions exercées par CCP5 pendant la spermatogenèse chez la souris. CCP5 est le seule enzyme qui a la capacité de couper le glutamate de branchement des chaines latéral et qui peut donc réguler l’équilibre entre présence ou l’absence de glutamate de branchement. L’analyse de la souris CCP5-knockout a permis de souligner le rôle essentiel mené par CCP5 pendant la spermiogénèse. J'ai constaté que les souris CCP5-KO produisent 100 fois moins de sperme, défectueux et immobile, comparé aux contrôles. De plus, des nombreuses cellules haploïdes immatures sont prématurément libérées de l’épithélium germinatif. Une analyse approfondie à révélée que la réduite production de sperme est due à plusieurs défaut ultrastructurelles qui surgissent pendant la spermiogenèse. J’ai observé que l’acrosome n’était pas bien développée et que cela se détachait du noyau chez les spermatides matures condensés. De plus l’organisation des microtubules formant la manchette était aussi affectée par une émanation ectopique, ainsi que par une localisation défectueuse dans le noyau. Ces défauts corrèlent avec la formation des spermatides allongée que n’ont pas la typique forme falciforme. De plus, j’ai constaté la présence de centrioles surnuméraires chez les spermatides allongées CCP5-KO. Ce défaut corrèle avec l’observation de microtubules « doublets » et « singlets » dispersés dans le cytoplasme de la cellule. De plus, les structures accessoires du flagelle se positionnaient, de façon désorganisé, à côté de ces microtubules. On a pu constater que ces microtubules sont très probablement issus de plusieurs axonemes qui s'ouvrent dans leur région. Le processus entier de spermiogenèse semble être défectueux dans la souris CCP5-KO et cela est accompagné par d'importants changements de niveaux de glutamylation chez les spermatides rondes et allongés. Par conséquence la régulation des niveaux de glutamylation faites par CCP5 lors de la spermiogénèse semble être fondamental pour garantir un développement normal des spermatides en spermatozoïdes. / Spermatogenesis is the process by which germ cells are transformed into spermatozoa by three sequential phases: the mitotic- and meiotic- phase followed by spermiogenesis. To allow the final maturation of haploid germ cells into spermatozoa specific structures have to be developed during the spermiogenesis: the acrosome, the manchette and the flagellum. The manchette is a MTs-based structure, located caudally to the acrosome, organizing in a skirt-like fashion. Manchette is known to participate in the shaping of the nucleus conferring it the typical hook-like shape and several studies have underlined its importance in acrosome and flagellum formation. During spermiogenesis all molecules and organelles necessary for both acrosome and flagellum formation have to be transported to their destination sites and manchettal MTs allow the movement of organelles and other proteins between the pro-acrosome region and the spermatid tail. However this MTs-based traffic has to be regulated both in space and time as it has been shown that ectopic or mislocalization of certain proteins can lead to failures in acrosome, manchette and flagellum development. The generation of posttranslationally modified MTs might explain a possible mechanism of traffic regulation since it has been demonstrated that posttranslational modifications (PTMs) can regulate the interaction between MTs and molecular motors and microtubules binding proteins. Polyglutamylation, consist in the addition of glutamate side chains of variable length on α- and β- tubulin carboxy-terminal tails. Glutamylation levels are determined by the combined action of glutamylase (TTLLs) and deglutamylase (CCPs) enzymes. Several reports have recently highlighted the importance of some of these enzymes in flagellum assembly and/or maintenance. During my PhD I investigated about the functional role of CCP5 during mouse spermatogenesis. CCP5 is the only enzyme able to remove the glutamate branching point of the added side chain. Thus, its activity might regulate the equilibrium between presence/absence of glutamate branching points, in turn interfering with polyglutamylation levels. The study of the CCP5-KO mouse reveals that CCP5 has an essential role during mouse spermiogenesis. CCP5-KO male produces 100-fold less sperm cells than controls and released sperm cells are highly defective and immotile. Moreover, haploid immature germ cells are also found in CCP5-KO semen. A deep-analysis reveals that the reduced sperm output is due to several ultrastructural defects emerging during the spermatids differentiation process. The acrosome, although is still formed, it does not appear to develop symmetrically and appears to detach from the nucleus in condensed spermatids. Another structure that is impaired in CCP5-KO spermatids in the manchette. Manchettal MTs, are seen to emanate from ectopic regions of the germ cells without running parallel to the nucleus, and are often observed within the spermatids nuclei. Altogether these defects correlate with an aberrant-shaped spermatid nucleus not showing the typical hook-like shape. Another phenotype observed in CCP5-KO elongating spermatids is the presence of supernumerary basal bodies that correlates with the presence of singlet or doublets microtubules dispersed within the germ cell cytoplasm. Interestingly sperm accessory structures are seen to chaotically organize around the microtubules. Unstable disassembling axonemes are seen together with those MTs, suggesting that CCP5-KO spermatids develop abortive unstable flagella. Interesting all these ultrastructural defects correlate with increased level of glutamylation on round spermatids’ cortical MTs and elongating spermatids’ manchettal MTs. Taken together, this study strongly suggests that CCP5-mediated glutamylation regulation is fundamental for spermatids differentiation into healthy functional spermatozoa.

Spermatogenesis is a highly orchestrated, strictly regulated cascade of events that could be divided into three major processes: mitotic expansion of diploid germ cells (spermatocytogenesis), meiotic division creating haploid cells, and spermiogenesis. Spermiogenesis, the final stage of spermatogenesis comprises a striking metamorphosis of round haploid spermatids into morphologically and functionally specialized spermatozoa designed for the fertilization. One of the proteins indispensable for proper sperm morphogenesis is centrobin, a structural component of the specialized cytoskeletal structures of the elongating spermatids (acroplaxome and manchette), executing essential role in sperm head shaping and assembly of the head-tail coupling apparatus. Disruption in Cntrob gene (coding for centrobin) in rats homozygous at the hd (hypodactyly) locus results in male infertility, with a striking morphological signature called "decapitated sperm syndrome" with detachment of sperm head from the flagellum due to impaired head-tail coupling. However, molecular function of centrobin in spermiogenesis is still unknown. Sperm decapitation is a distinct phenotype described in several mouse mutants and importantly from infertile human males. Strikingly, in addition to proteins functioning in cytoskeletal...

Testicular maturation indices (TMI) were determined for wild-caught males of Poecilia mexicana by quantitative analysis of the frequency of ongoing stages of spermatogenesis and spermatid differentiation in sections of testes stained with the Feulgen reaction for DNA. In nature, males maintain essentially constant levels of sperm production throughout the year and show no significant variations in mean TMI values associated with season, microhabitat, standard body length, or rank in male dominance hierarchies. Winter males or males isolated from females in the laboratory show accumulation of mature spermatophores. These findings suggest that fluctuations in the microstructure of wild populations of Poecilia from northeastern Mexico may be due primarily to differences in the temporal scheduling of female reproductive cycles and not to limitations imposed by sperm availability or male reproductive competence. The data are discussed in terms of competitive interactions within unisexual-bisexual breeding complexes involving the Amazon molly P. formosa, its related triploid hybrids, and the bisexual species, P. mexicana.