The multilayered structure (MLS), a component of the locomotory complex of plant sperm, has been utilized extensively by taxonomists in establishing phylogenetic relationships between the lower plants and algae. Unfortunately, there has been almost no biochemical characterization of the MLS and, in those studies that did attempt a characterization, conflicting results were obtained. We utilized antisera to the calcium-binding protein centrin to probe thin sections of the mid-stage spermatids of the anthocerote Phaeoceros laevis, the hepatic Sphaerocarpos texanus, and the pteridophyte Ceratopteris richardii embedded in L. R. White resin. The lamellar strip (LS; layers S2-S4) of the MLS in each of these species is labelled strongly with anti-centrin, but the S1 layer, composed of microtubules, is not. In Ceratopteris, centrin is also detected in the amorphous electron opaque material that connects the basal bodies of the flagella. Both the MLS and the amorphous zones are putative microtubule organizing centers. Extracts from axenic cultures of Ceratopteris subjected for electrophoresis and Western blotting revealed a reactive band at 19.3 kDa, a protein similar in molecular mass to algal centrin. These data indicate that the MLS is composed at least partially of the protein centrin or a protein antigenically-related to centrin. This report is the first electron microscopic immunocytochemical demonstration that a centrin homologue is found in land plants and that it occurs at putative microtubule organizing centers.

Submitted by Emi Rosane Silistino de Souza null (emi.rosane@hotmail.com) on 2016-03-10T05:51:12Z No. of bitstreams: 1 DISSERTAÇÃO EMI 2016 OK.pdf: 3134204 bytes, checksum: c2496577f07709557fe6c8ab1c101ffb (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-03-10T18:53:23Z (GMT) No. of bitstreams: 1 souza_ers_me_sjrp.pdf: 3134204 bytes, checksum: c2496577f07709557fe6c8ab1c101ffb (MD5) / Made available in DSpace on 2016-03-10T18:53:23Z (GMT). No. of bitstreams: 1 souza_ers_me_sjrp.pdf: 3134204 bytes, checksum: c2496577f07709557fe6c8ab1c101ffb (MD5) Previous issue date: 2016-03-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A Subordem Heteroptera contém cerca de 75 famílias, incluindo Coreidae, na qual pertencem as espécies Corecoris fuscus, Leptoglossus gonagra, L. zonatus e Sphictyrtus fasciatus, analisadas no presente trabalho. Os indivíduos dessa família são conhecidos por serem exclusivamente fitófagos e alimentam-se de plantas de valor comercial. Apesar da grande diversidade desta subordem, há poucos estudos relacionados com a ultraestrutura da espermátide dessas espécies. Assim, nesse trabalho, por meio da microscopia eletrônica de transmissão, foi analisado o desenvolvimento da espermiogênese na cabeça e no flagelo, com ênfase na formação do acrossomo, do adjunto do centríolo e dos derivados mitocondriais. O acrossomo tem origem a partir do complexo de Golgi, por meio da formação de uma vesícula pró-acrossomal e concomitante a esse processo é formado o adjunto do centríolo. O acrossomo determina o polo anterior do espermatozoide e o adjunto do centríolo determina o polo posterior. Observou-se também que no início do processo espermiogênico, as mitocôndrias encontram-se espalhadas no citoplasma, então gradualmente se fundem e dão origem ao complexo mitocondrial, o qual se divide, posteriormente, em duas subestruturas, denominadas derivados mitocondriais. Essas se posicionam bilateralmente ao axonema, o qual possui o padrão típico de disposição dos microtúbulos de 9 + 9 + 2. O processo de formação do acrossomo e dos derivados e a conformação dos microtúbulos do axonema são típicos de insetos. Além do padrão de microtúbulos do axonema, foram encontradas características sinapomórficas de Heteroptera, como a presença de duas estruturas paracristalinas em cada derivado mitocondrial e a presença de pontes que ligam os derivados mitocondriais ao microtúbulos axonemais, os quais são ferramentas importantes para auxiliar análises filogenéticas e taxonômicas. / The suborder Heteroptera contains about 75 families, including Coreidae, to which the species Corecoris fuscus, Leptoglossus gonagra, L. zonatus and Sphictyrtus fasciatus belong. Individuals of this family are known for being exclusively phytophagous and feeding on commercially valuable plants. Despite this suborder great diversity, there are only few studies on the ultrastructure of these species spermatid. Thus, in this study, through transmission electron microscopy, we analyzed the development of spermiogenesis in the head and the flagellum of C. fuscus, L. gonagra, L. zonatus and S. fasciatus, with emphasis on the formation of the acrosome, of the centriole adjunct and on development of mitochondrial derivatives. The acrosome originates from the Golgi complex through the formation of a pro-acrosomal vesicle and concomitant to this process, is formed the centriole adjunct. The acrosome determines the anterior pole of the sperm and the centriole adjunct determines the posterior pole. It was also observed that at the beginning of the spermiogenic process, the mitochondria are scattered within the cytoplasm, then gradually fusing and originating the mitochondrial complex, which is subsequently divided into two substructures, called mitochondrial derivatives. They are positioned bilaterally to the axoneme, having the typical pattern arrangement of the microtubule, which is 9 + 9 + 2. The process of acrosomes and derivatives formation and the conformation of axoneme’s microtubules are typical of insects. Besides the pattern of axoneme’s microtubules, synapomorphic features of Heteroptera were found, as the presence of two paracrystalline structures in each mitochondrial derivative and the presence of bridges connecting derivatives mitochondrial to the axonemal microtubules, which are important tools to assist in phylogenetic and taxonomic analyzes. / FAPESP: 2013/19864-5

During spermatogenesis, histones are largely replaced by transition proteins and protamines in normal stallions. Incomplete nucleoprotein exchange results in the abnormal retention of histones and transition proteins, which is an indicator of poor sperm quality. Equine nucleoprotein exchange has not previously been investigated in detail, so that equine sperm chromatin quality problems, which are often responsible for poor breeding performance of stallions, are not well understood. In order to characterize chromatin remodeling events in stallion spermatogenesis and to identify proteins indicative of sperm chromatin defects, such as excessive amounts of histones, we identified antibodies that recognize equine testis-specific proteins of interest. Immunoblotting of testis and sperm protein lysates and immunofluorescence staining of histological tissue sections were used to identify candidate marker proteins of incomplete sperm chromatin maturation. Results of the study, which represents the first comprehensive characterization of the nucleoprotein exchange during spermatogenesis in the stallion, challenge the paradigm that the main function of histone H4 lysine (hyper-) acetylation (concomitant H4K5 and H4K8 acetylation) is to facilitate nucleosome ejection during spermatid nuclear elongation to allow for transition protein and protamine insertion into the chromatin. That paradigm was based on observations in mice and rats where H4 acetylation in several lysine residues occurs just prior to or during nuclear elongation. In contrast, the equine data presented here show strong acetylation of H4 in K5, K8 and K12 positions immediately after meiosis in round spermatids, independent of nuclear transition protein 1 deposition. Furthermore, results of H4K16 acetylation analyses underline the importance of this mark, which is likely mediated by DNA damage signaling pathways, emphasizing the importance of DNA repair processes for the exchange of nucleoprotein exchange in spermiogenesis and therefore, in extension, for male fertility. In addition, a revised description of the equine spermatogenic cycle is proposed here that is better aligned with human, mouse and rat spermatogenesis. Finally, the testis-specific histone variant TH2B was identified as a potential quantitative marker of equine sperm quality.

No description available.

My degree work focused on the problem of involuntary childlessness, which is a situation that a number of couples are facing nowadays and this issue has been growing. The work specifically deals with the donor programme in assisted reproduction. I described the method of treatment with the use of donor gametes and ethical and legislative issues related to it. The empiric part of the work is divided into two research projects each of which corresponds with one objective of the work. The first one monitors the situation when receiving couples are making a decision whether to join the assisted reproduction programme with donor gametes and the mental and social consequences of this situation. This research part is based on partly structured and non-structured interviews with 10 couples and on observations. The data was transcribed and subsequently qualitatively analysed. The second research comprises data obtained from non-structured interviews with female donors and from on-site observations. This part relates to the second objective of the degree work. In the discussion section of the work the interviews and observations are assessed and compared with literature on this topic. I focused mainly on the issue of anonymous donation, on motivation of oocyte donors and on subsequent comparison with receivers´ expectations. Based on the research I determined a hypothesis that donation of gametes is a difficult problem which our society should deal with in detail. This degree work could be used by people from centres of assisted reproduction. It can also be used as a study material by students of the Faculty of Health and Social Studies, especially those who attend the midwife course.

Résumé : La phase haploïde de la spermatogenèse (spermiogenèse) est caractérisée par une modification importante de la structure de la chromatine et un changement de la topologie de l’ADN du spermatide. Les mécanismes par lesquels ce changement se produit ainsi que les protéines impliquées ne sont pas encore complètement élucidés. Mes travaux ont permis d’établir la présence de cassures bicaténaires transitoires pendant ce remodelage par l’essai des comètes et l’électrophorèse en champ pulsé. En procédant à des immunofluorescences sur coupes de tissus et en utilisant un extrait nucléaire hautement actif, la présence de topoisomérases ainsi que de marqueurs de systèmes de réparation a été confirmée. Les protéines de réparation identifiées font partie de systèmes sujets à l’erreur, donc cette refonte structurale de la chromatine pourrait être génétiquement instable et expliquer le biais paternel observé pour les mutations de novo dans de récentes études impliquant des criblages à haut débit. Une technique permettant l’immunocapture spécifique des cassures bicaténaires a été développée et appliquée sur des spermatides murins représentant différentes étapes de différenciation. Les résultats de séquençage à haut débit ont montré que les cassures bicaténaires (hotspots) de la spermiogenèse se produisent en majorité dans l’ADN intergénique, notamment dans les séquences LINE1, l’ADN satellite et les répétions simples. Les hotspots contiennent aussi des motifs de liaisons des protéines des familles FOX et PRDM, dont les fonctions sont entre autres de lier et remodeler localement la chromatine condensée. Aussi, le motif de liaison de la protéine BRCA1 se trouve enrichi dans les hotspots de cassures bicaténaires. Celle-ci agit entre autres dans la réparation de l’ADN par jonction terminale non-homologue (NHEJ) et dans la réparation des adduits ADN-topoisomérase. De façon remarquable, le motif de reconnaissance de la protéine SPO11, impliquée dans la formation des cassures méiotiques, a été enrichi dans les hotspots, ce qui suggère que la machinerie méiotique serait aussi utilisée pendant la spermiogenèse pour la formation des cassures. Enfin, bien que les hotspots se localisent plutôt dans les séquences intergéniques, les gènes ciblés sont impliqués dans le développement du cerveau et des neurones. Ces résultats sont en accord avec l’origine majoritairement paternelle observée des mutations de novo associées aux troubles du spectre de l’autisme et de la schizophrénie et leur augmentation avec l’âge du père. Puisque les processus du remodelage de la chromatine des spermatides sont conservés dans l’évolution, ces résultats suggèrent que le remodelage de la chromatine de la spermiogenèse représente un mécanisme additionnel contribuant à la formation de mutations de novo, expliquant le biais paternel observé pour certains types de mutations. / Abstract : Germline mutations may arise from several endogenous and exogenous mechanisms in both male and female. However, recent next-generation sequencing (NGS) data confirmed that de novo mutations arise primarily in males. This observation suggests that specific spermatogenesis events are involved in the male mutation bias. One potential origin for male-driven mutations is the differentiation of spermatids into spermatozoa, which involves one of the most striking and global chromatin remodeling processes, where histone-bound chromatin is converted into highly condensed protaminated DNA toroid. Using pulse-field gel electrophoresis and comet assay on flow cytometry sorted cells, it was established that chromatin remodeling process is characterized by a transient surge in DNA double strand breaks (DSBs) in the whole population of murine spermatids, which get repaired by the end of spermiogenesis. Using a highly active nuclear extract and immunofluorescences, topoisomerases and markers of DNA repair systems were shown at these steps. Since haploid cells cannot rely on homologous recombination for templated DNA repair, it was hypothesized that this process may be genetically unstable and largely responsible for the observed male de novo mutations bias. Although very challenging, a method allowing the specific genome-wide mapping of DSBs using NGS was developed to establish the genomic distribution of DSBs during chromatin remodeling. It was shown that intergenic regions were enriched in DSBs, particularly LINE1, satellite DNA and simple repeats. Motif finding on potential hotspots showed that proteins from FOX and PRDM families may be implicated. Although homologous recombination cannot take place during spermiogenesis, an enrichment in BRCA1 motif was found, which is also known to be implicated in NHEJ and removal of topoisomerase adducts. Topoisomerase-like SPO11 motif was also enriched suggesting that the meiotic machinery may also be implicated during chromatin remodeling. Moreover, although DSBs tend to accumulate in intergenic regions, gene ontology analysis of hotspot-containing genes showed a marked enrichment in genes related to neurons and brain development. This result hence supports the fact that neurological disease associated mutations are also male biased and associated with advanced paternal age. Since DSB formation during spermiogenesis is conserved through evolution, these results suggest that chromatin remodeling in spermatids represents a significant component in the reported male de novo mutation bias.