Defects in meiotic prophase I events, resulting in aneuploidy, are a leading cause of birth defects in humans; however, these are difficult to study in mammalian systems due to their occurrence very early in development. The nematode, Caenorhabditis elegans, is an excellent model for prophase I studies as its gonad is temporally and spatially organized around these meiotic events. Homolog pairing, synapsis, meiotic recombination and crossover formation are essential to the proper segregation of chromosomes into the respective gametes, either the egg or sperm. Disturbances in these events leads to missegregation of chromosomes in the gametes in the meiotic divisions. Synapsis is especially critical in meiosis as it precedes and is required for meiotic recombination in C. elegans. The formation of the synaptonemal complex (SC) is fundamental to chromosomal synapsis, yet the molecular mechanisms of synaptonemal complex morphogenesis are largely unknown. The investigations described in this thesis were undertaken to better understand the molecular contributions to synaptonemal complex morphogenesis. Chapter One reviews knowledge of morphogenesis and its relationship to the events of meiotic prophase I. Recent studies in our laboratory have implicated AKIRIN, a nuclear protein with multiple biological functions, as having a role in synaptonemal complex disassembly, specifically preventing the aggregation of synaptonemal proteins (Clemons et al., 2013). As a result of our efforts to discern the mechanism by which AKIRIN regulates disassembly, we found that the highly conserved CSN/COP9 signalosome has a role in SC assembly, leading to defects in prophase I events and in MAPK signaling , leading to the arrest of nuclei in the later stages of meiosis. While the CSN/COP9 signalosome has been implicated in general fertility in C. elegans (Pintard et al., 2003), no role had been defined in earlier meiotic stages until this study. Chapter Two describes an RNAi enhancer/suppressor screen undertaken in the akir-1 mutant background. Several RNAi clones were selected for future study based on a reduction in brood size; one of which, csn-5/, is the focus of the analysis presented in Chapter 3. Chapter Three describes the phenotypic characterization of two CSN/COP9 signalosome subunits, csn-2 and csn-5. Alleles of both genes display synaptonemal complex protein aggregation and defects in mitotic cell proliferation, homologous chromosome pairing, meiotic recombination and crossover formation, leading to an increase in apoptosis. Oocyte maturation is also disrupted by a lack of MAPK signaling, resulting in a lack of viable oocytes, which renders the csnmutant homozygotes sterile. These findings support a model suggesting the CSN/COP9 signalosome has an essential role in regulating meiotic prophase I events and oocyte maturation. Chapter 4 describes the methodology used in this study. Chapter 5 provides a summary of the thesis findings and examines the future directions to extend this work.

Abstract not available

AN ABSTRACT OF THE DISSERTATION OF RENEE A LOPEZ SWALLS, for the Doctor of Philosophy degree in PLANT BIOLOGY, presented on June 27, 2016, at Southern Illinois University Carbondale. TITLE: THE SPECIAL WALLS AROUND GAMETES IN CERATOPTERIS RICHARDII AND AULACOMNIUM PALUSTRE: USING IMMUNOCYTOCHEMISTRY TO EXPOSE STRUCTURE, FUNCTION, AND DEVELOPMENT MAJOR PROFESSOR: Dr. Karen S. Renzaglia Gametes are arguably the most important cells formed during the sexual life cycle of plants. The ancestral condition of gametes in land plants is the production of small motile sperm cells and larger non-motile eggs. Unique walls or cell matrices are formed during the development of these highly specialized cells, and are integral to their proper development and maturation. Yet, the polysaccharide composition, structural function, and metabolic processes of these special gamete cell walls remain unexplored beyond examination in the light microscope. Utilizing histochemical techniques coupled with immunocytochemical localizations with monoclonal antibodies (MAbs), I give a detailed survey of AGP and cell wall polymer distribution during male and female gametogenesis in the model fern, Ceratopteris richardi, and for comparison with both Ceratopteris and seed plants, I examined the same cell wall polymers during spermatogenesis in Aulacomnium palustre, a moss species. AGPs are abundant in the extraprotoplasmic matrix that surrounds differentiating sperm and egg cells in the fern, Ceratopteris richardii. During spermatogenesis, AGPs are speculated to regulate growth of flagella and cell morphogenesis through cell signaling via Ca+2 oscillations. Immunogold localizations revealed that AGPs are differentially expressed in the egg envelope in C. richardii. These glycoproteins are extremely abundant prior to fertilization but decrease substantially after fusion of the male gamete with the egg cytoplasm. Contrary to the AGP-filled matrix surrounding developing spermatids, developing eggs are bathed in (1,5)-α-L-arabinan pectins and not AGPs. Lastly, I examined the unique cell walls that are integral to sperm cell differentiation and release in both Ceratopteris and Aulacomnium. The preponderance of callose and hemicelluloses in the walls of the male gametes of Ceratopteris and Aulacomnium, respectively, and the importance of these polysaccharides in development are discussed. Taken together, the studies that comprise this dissertation advance significantly our understanding of cell wall dynamics during gametogenesis in early land plants.

Orientadores: Sandra Maria Carmello-Guerreiro, Simone de Padua Teixeira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T03:29:24Z (GMT). No. of bitstreams: 1 Cortez_PriscilaAndressa_M.pdf: 142634667 bytes, checksum: 533aad1a400f13a4d678be944c37b495 (MD5) Previous issue date: 2007 / Resumo: Miconia albicans (Melastomataceae) mostrou-se uma espécie com agamospermia obrigatória do tipo diplosporia e viabilidade polínica nula em populações do Cerrado e da Floresta Atlântica. A inviabilidade polínica não esteve restrita a um único estádio de desenvolvimento dos andrófitos e a principal anormalidade observada nas anteras de flores em pré-antese foi a deformidade das células reprodutivas, principalmente aquelas em estádio de andrósporos livres. A intensa vacuolação das células androsporogênicas e tapetais, bem como a precoce degeneração das células do tapete, podem ser características relacionadas à esterilidade masculina da espécie. A ontogenia dos estratos parietais diferiu da observada em outros membros do gênero, corroborando a sua classificação como irregular. Anteras em estádio de pré-deiscência apresentaram a região de deiscência coberta por uma provável epiderme com características distintas daquela observada no restante da mesma. Diferenças ambientais foram observadas apenas na quantidade de células encontradas nas anteras de flores em pré-antese, cerca de 50% maior nos indivíduos provenientes do Cerrado. A ontogenia do óvulo e do ginófito se assemelhou àquela observada em outras espécies do gênero, exceção feita a divisão da célula arquesporial e a supressão da meiose em M. albicans. Os embriões agamospérmicos foram originados no interior do ginófito, a partir da oosfera ou de uma das duas sinérgides. Evidências sugerem que o endosperma autônomo do tipo nuclear foi originado por expansões de cada um dos núcleos da célula central, e não por sua fusão. A porcentagem de aborto de óvulos na espécie foi de cerca de 50% e as sementes maduras produzidas apresentaram uma porcentagem de germinação bastante variável, provavelmente em decorrência da alta incidência de fungos observada nos experimentos realizados. Os frutos formados em botões florais apresentaram características semelhantes às observadas nas demais unidades, incluindo a viabilidade das sementes / Abstract: Miconia albicans (Melastomataceae) is a species with diplosporic obligate agamospermy and no pollen viability in ¿Cerrado¿ and Atlantic Forest vegetations. The sterility of pollen grains wasn¿t restricted to one developmental stage and the major abnormality of the cells from pre-anthesis flower anthers was the deformation of the reproductive cell wall, mainly in the free microspore stage. Intense vacuolation of both sporogenous and tapetal cells as well as the precocious degeneration of the tapetal cells may be related to male sterility in this species. The ontogeny of the parietal layers was different from that observed in another species of the genus and was in agreement with its classification of irregular type. The anthers in predehiscence stage showed the dehiscence region probable closed with an epidermis with papilous cells, differently from that observed in the another anther regions. Ambient differences were restricted to the number of cells observed in the pre-anthesis anthers, which was about 50 percent up in the Atlantic Forest vegetation in relation to the Cerrado one. The ovule and embryo sac ontogeny was similar to that observed in another species of the genus, except for the division of the arquesporic cell and the suppression of the meiotic division during the gametogenesis, both observed only in M. albicans. The agamospermous embryos were originated in the embryo sac from the egg cell or one of two synergids. Evidence suggested that the nuclear autonomous endosperm was originated from expansions in both nucleus of the central cell, and not from the fusion of both. The percentage of ovule abortion was about 50 percent and the mature seeds showed a variable germination percentage probably due to fungi incidence which occurred in the seeds from both environments. The ontogenetic characteristics observed in fruits from both floral buds and flowers were similar, as well as the seeds viability / Mestrado / Biologia Vegetal / Mestre em Biologia Vegetal

Orientadores: Sandra Maria Carmello-Guerreiro, Simone de Padua Teixeira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T16:53:33Z (GMT). No. of bitstreams: 1 Caetano_AnaPauladeSouza_M.pdf: 5552395 bytes, checksum: ddd2d3795d5da107ef405fe68e7a02a3 (MD5) Previous issue date: 2010 / Resumo: Em Miconia fallax, uma espécie apomítica, e Miconia pepericarpa, uma espécie sexuada, os processos de esporogênese e gametogênese apresentam diversas características semelhantes, muitas delas compartilhadas com outros membros da família Melastomataceae. Entretanto, apesar das similaridades, algumas diferenças foram observadas. Em relação à antera, o formato da célula generativa no grão de pólen maduro é fusiforme em M. pepericarpa e esférico em M. fallax. Além disso, a quantidade de grãos de pólen viáveis produzidos é marcadamente menor na espécie apomítica. Quanto ao óvulo, as diferenças encontradas são ainda mais marcantes. Em M. fallax, células denominadas iniciais apospóricas se diferenciam juntamente com as células-mãe de megásporos, levando à formação de mais de um saco embrionário. Assim, enquanto M. fallax apresenta até três sacos embrionários no óvulo maduro, sendo um sexuado e até dois apospóricos, M. pepericarpa exibe apenas um saco embrionário. Além disso, na espécie apomítica M. fallax, constatou-se a formação de embriões adventícios em sementes jovens. Provavelmente, o desenvolvimento de embriões a partir dos sacos embrionários apospóricos juntamente com a formação do embrião zigótico e adventício levam à poliembrionia em M. fallax, com formação de até quatro embriões em uma mesma semente. Das diferenças encontradas entre as duas espécies, apenas o formato da célula generativa não foi relacionado à apomixia, sendo um caráter específico. Já as demais diferenças, são características exclusivas de M. fallax, e estão diretamente relacionadas à apomixia, sendo elas: 1) formação de uma proporção menor de grãos de pólen viáveis; 2) diferenciação de células iniciais apospóricas; 3) formação de múltiplos sacos embrionários; 4) desenvolvimento de embriões apomíticos; 5) poliembrionia. A espécie apomítica M. fallax é poliplóide e, neste caso, a viabilidade polínica baixa pode ser explicada pela ocorrência de alterações na meiose, comuns em espécies poliplóides. A poliembrionia, também tem sido bastante relacionada à apomixia, sendo esta relação confirmada pelos dados obtidos neste trabalho. Além disso, com os resultados alcançados, foi possível reconhecer que M. fallax é uma apomítica facultativa, uma vez que, além da possibilidade de produção de embriões apomíticos, a espécie ainda preserva os mecanismos de formação de grãos de pólen e sacos embrionários sexuados, com possibilidade de produção de embriões zigóticos. / Abstract: In Miconia fallax, an apomictic species, and Miconia pepericarpa, a sexual species, the processes of gametogenesis and sporogenesis have a number of similar characteristics, many of which are shared with other members of the family Melastomataceae. However, despite the similarities, some differences were observed. Concerning the anther, the generative cell in mature pollen grain is spindle shaped in M. pepericarpa and spherical in M. fallax. Furthermore, the quantity of viable pollen grains produced is markedly lower in apomictic species. Regarding the ovule, differences are even more striking. In M. fallax, cells called aposporous initials differentiate themselves along with the megaspore mother cell, leading to the formation of more than one embryo sac. Thus, while M. fallax exhibit up to three embryo sacs in mature ovule, one being sexed and a maximum of two aposporous, M. pepericarpa displays only one embryo sac. Moreover, in apomictic species M. fallax, we found the formation of adventitive embryos in young seeds. Probably the development of embryos from the aposporous embryo sac parallel to the formation of zygotic and adventitious embryos lead to polyembryony in M. fallax, with formation of up to four embryos in a single seed. About differences found between this two species, only the shape of the generative cell was not related to apomixis, as it is a specific character. The other differences are unique features of M. fallax and are directly related to apomixis, namely: 1) the formation of a smaller proportion of viable pollen grains, 2) the differentiation of aposporous initial cells 3) the formation of multiple embryo sacs, 4) the development of apomictic embryos (adventitious or aposporic), and 5) polyembryony. The apomictic species M. fallax is polyploid, and, in this case, the low pollen viability can be explained by the occurrence of changes in meiosis, common in polyploid species. The polyembryony, has also been frequently related to apomixis, and this relationship is confirmed by data obtained in this work. Furthermore, with these results, it was possible to recognize that M. fallax is a facultative apomictic, since in addition to the possibility of apomictic embryo production the species still preserves the mechanisms of the formation of pollen grain and sexual embryo sac, with the possibility of producing zygotic embryos. / Mestrado / Mestre em Biologia Vegetal

Sexual reproduction in Porites divaricata (Le Sueur, 1820) was studied over a one year period (August, 2012 to August, 2013) in a shallow water (~2m depth) carbonate bank (also described as marginal) habitat at Rodriguez Key, a small island located 1.44 km off the east coast of Key Largo. Porites divaricata is a brooding, gonochoric species with peak reproductive output (planula release) occurring in March and at least a smaller event in May. This species showed an overall female to male sex ratio of 3.5:1. However, during peak reproduction, the sex ratio was 1.2:1. Spawning (sperm release) most likely occurred in late February when 78% of all oocytes and 84% of all spermaries were in stage IV of development. The largest output of stage IV eggs (n=164) occurred at this time, and these oocytes also had the second largest average individual oocyte volume (3.34x10-3 mm3, n = 79) compared to those from other sample dates. The largest average egg volume (3.79x10-3 mm3, n = 8) occurred in May, but with fewer eggs present (n=13). Population surveys showed P. divaricata densities of 7.4 and 17.7 colonies m-² at Site 1 and 2, respectively. Temperature data documented that SSTs were highly variable in the seagrass/coral habitat at site 2 (primary collection site), ranging from 13.1 to 34.9°C, with an average temperature of 25.9°C. Corals at Rodriquez Key are exposed to temperatures much higher (+3.8°C) during the summer and much lower (-8.2°C) in winter compared to corals living at a nearby bank reef (Molasses Reef). This is the first comprehensive reproductive study performed on Porites divaricata and may provide valuable information to the ongoing debate concerning the taxonomic relationships among P. divaricata, P. porites, and P. furcata.

Le paludisme est causé par des parasites du genre Plasmodium et la gamétogenèse est une étape essentielle à sa transmission. Afin d'améliorer les connaissances sur la genèse des gamètes, nous avons mené des études protéomiques sur des gamétocytes de P. berghei sauvages ou mutants avec le gène moteur de la kinésine 8 interrompu. Le mutant est morphologiquement semblable au type sauvage mais incapable de compléter la gamétogenèse et les gamètes mâles d'exflageller. A partir d'échantillons enrichis en gamétocytes, la gamétogenèse a été suivie sur 15 min après induction par l'acide xanthurénique. Les peptides marqués par iTRAQ à partir de triplicatas biologiques ont été analysés par nanoLC/MS-MS et interprétés par les logiciels Patternlab et Blast2GO. Les phosphopeptides ont été enrichis au TiO2. Chez le parasite sauvage, 443 protéines et 206 phosphoprotéines ont été identifiées à partir de 2617 peptides et chez le mutant, 530 protéines et 218 phosphoprotéines à partir de 3198 peptides. L'induction de la gamétogenèse est marquée par une transcription, une biosynthèse de protéines et de l'ADN importantes. Chez le mutant, la formation des fuseaux mitotiques et des axonèmes des flagelles des gamètes mâles est fortement affectée. Des protéines du complexe du protéasome dépendant de l'ubiquitine, de la réponse au stress, du métabolisme énergétique sont également affectées. Cette étude apporte de nouveaux éléments de compréhension sur le rôle joué par la kinésine 8 dans la gamétogenèse de Plasmodium. / Malaria is caused by parasites from Plasmodium genus and its gametogenesis is an essential step to ensure the malaria transmission. In efforts to improve the knowledge on gamete biology we did quantitative proteomic and phosphoproteomic comparative experiments with P. berghei WT and gametocytes disrupted for the motor gene kinesin 8. The mutant is morphologically similar to wild-type parasite but impaired gametogenesis and unable to exflagellate. The Gametocytes-enriched sample from mice at time T0 were xanthurenic acid-induced to exflagellate and harvested at time T7 and T15. The iTRAQ labelled peptides from independent biological triplicate sample were analyzed overtime through nanoLC/MS-MS Orbitrap after TiO2 enrichment, and interpreted by Patternlab and Blast2GO softwares. The wild type had 443 proteins and 206 phosphoproteins identified from 2,617 peptides. The mutant had 530 proteins and 218 phosphoproteins identified from 3,198 peptides. GO biological processes related to RNA translation, DNA and protein biosynthesis were most prominent and phosphorylated proteins are mainly RNA, ATP or protein binding proteins. Within the mutant, the axoneme and mitotic spindle microtubules disorganization were strongly affected. The nucleosome components are key to nuclear division disorganization. The ubiquitin-dependent proteasome complex and stress/folding response, energy metabolism and egress proteins were affected. The Plasmodium proteomic approach brings that insight into kinesin 8 critical importance for male gametogenesis in Plasmodium with effect on protein expression, phosphorylation modulation, flagellar organization and biology.

Embryonic chromatin diminution, the selective excision of large amounts of heterochromatic DNA from presomatic cell lineages, provides an example of an unusually large augmentation of the germline genome and raises questions regarding the source of the increased amount of DNA and its relevance to the biology of the organism. DNA levels in adult germ cell nuclei of the copepod Cyclops kolensis were determined by DNA-Feulgen cytophotometry and compared with those of somatic nuclei of adults and both pre- and postdiminuted embryos from the same mothers. Almost 75 pg DNA/nucleus is excised by diminution, resulting in the return of each generation to the approximately 1 pg DNA/nucleus level found for adult soma. To account for the increase in DNA levels of germ cells observed here, we propose alternative hypotheses to the original model of chromatin diminution: (1) repetitive endocycles or (2) proliferation of genetic elements. Specific tests for these hypotheses using next-generation sequencing and quantitative cytophotometry, as well as the functional significance of germ cell DNA augmentation to the copepod, are discussed.

The decline of many freshwater mussel populations in the United States has brought about the need for facilities in which mussels can be held for purposes of relocation, research, and propagation. The Aquatic Wildlife Conservation Center (AWCC) of the Virginia Department of Game and Inland Fisheries (VDGIF) serves as a freshwater mussel conservation facility in southwest Virginia. The goals of this study were: (1) to determine whether adult freshwater mussels could maintain energy reserves at AWCC (2) to determine whether adults could produce mature gametes at AWCC and (3) to establish suitable rearing conditions for juvenile mussels at the AWCC. In fall 2002, four species of mussels, Villosa iris, V. vanuxemensis, Amblema plicata, and Pleurobema oviforme, served as surrogates for endangered species and were relocated to the AWCC. Three energy reserves (glycogen, protein, and lipid) were measured seasonally (fall 2002 to summer 2004) from mantle tissue and compared between AWCC specimens and those from their wild source populations. The gametogenic stage of each species was also compared to determine whether gametogenesis was occurring in captivity. In summer 2003, the first of two juvenile experiments tested the effects of three rates of water flow (1 L/min, 3 L/min, and 7 L/min) on the survival and growth of V. iris and Epioblasma capsaeformis reared in flow-through troughs. In summer 2004, round flow-through tanks were used to assess the effects of three sizes of substrate (fine sediment, fine sand, and coarse sand) and sampling frequency on the survival and growth of V. iris. Gut content analyses also were conducted at the end of each experiment to determine which algal species were being consumed. Overall survival rates were as follows: A. plicata, 100 %; V. vanuxemensis, 86 %; V. iris, 79 %; P. oviforme (2002 collection), 53 %; and P. oviforme (2003 collection), 50 %. All energy reserves varied among seasons, but every species except P. oviforme (2003 collection) had levels higher than those in source populations at the end of this experiment. Glycogen appeared to be the best indicator of condition in these species, with protein also being important in the 2003 collection of P. oviforme. Mature gametes were found in all four captive species in 2003 and 2004, with lipids appearing to fuel gametogenesis. Additionally, gametogenesis was occurring earlier in captive long-term brooders than in the wild, possibly due to warmer water temperatures at AWCC. The first juvenile experiment resulted in 15 % overall survival, with 1 L/min having the greatest survival (18 %), and the 3 L/min having the greatest growth (656 μm). In the second experiment, dishes left unsampled had significantly greater survival (40 %) (P<0.05) of juveniles than those which were sampled (27 %). The unsampled fine sand treatment had significantly greater survival than the other two unsampled treatments (52 %) (P<0.001). Sampled juveniles in fine sediment had the greatest growth (887 μm). Also, juveniles from Experiment 1 were consuming primarily Navicula, with Coelastrum and Chlorella consumed in greatest abundance in Experiment 2. Results indicate that most adult mussels maintained energy reserves and produced mature gametes, and that juveniles of V. iris had good survival and growth. Only P. oviforme had survival rates lower than expected and did not appear to maintain condition at AWCC. Based on results of the species tested, environmental conditions at AWCC appear suitable for the survival of most adult and juvenile freshwater mussels. / Master of Science

Due to the increasing need to provide refugia for freshwater mussels impacted by anthropogenic activities and exotic species, facilities should be identified and protocols developed for holding mussels in captivity. White Sulphur Springs National Fish Hatchery (WSSNFH), White Sulphur Springs, WV, has held freshwater mussels for nearly eight years, and has the potential to become an important refugium and propagation facility for conservation of mussels in the Ohio River Basin and elsewhere. The goal of this study was to determine the feasibility of holding adult freshwater mussels in long-term captivity at WSSNFH by evaluating survival, energy reserves, and gametogenesis of captive mussels in a recirculating pond system. I relocated three mussel species in the summer of 2001 and 10 mussel species in the summer of 2002 to a recirculating pond system (reservoir and raceway) at the hatchery. Water quality parameters of pH, alkalinity, hardness, temperature, and dissolved oxygen; and algal concentrations were measured periodically from summer 2001 to summer 2003. Annual survival rates of 10 species were estimated (August 2002 to August 2003) using the program MARK. Glycogen, protein, and lipid concentrations in mantle tissue of three captive species (Actinonaias ligamentina, Cyclonaias tuberculata, and Tritogonia verrucosa) were compared to those of wild mussels in the New River. Gametogenic activity and synchrony in A. ligamentina and C. tuberculata were compared between captive and wild mussels. Water quality parameters, with the exception of temperature, were within desirable ranges for most of the study. Temperatures of > 28° C were observed for several days during summers 2002 and 2003. Algal concentrations averaged 1903 cells ml-1 in the raceway (range: 300 to 4658 cells ml-1), which is comparable to algal concentrations reported for nearby rivers. The overall survival rate for 10 freshwater mussel species held in the raceway for one year was 77%. Villosa vanuxemensis had the highest survival rate (96%), and Lampsilis cardium had the lowest survival rate (31%). Although there were fluctuations in glycogen, protein, and lipid levels over 2 yr, there were no overall differences in energy substrates between captive and wild mussels at the end of the study. Captivity did not appear to have a negative affect on gametogenesis. Captive C. tuberculata spawned within the expected time frame between January and June, but slightly earlier than their wild counterparts in the New River. Due to the infestation of the gonads of both captive and wild A. ligamentina by digenean trematodes, little gametogenesis was observed. However, captive holding did not appear to have an effect on trematode infestation rates. From these results, I conclude that captive holding conditions in the recirculating pond system at WSSNFH were adequate for long-term holding of a wide range of mussel taxa. I recommend that WSSNFH continue to be used as an adult holding facility. Further research should be conducted to determine food and habitat preferences of freshwater mussel species in captivity so that optimal holding conditions can be provided for each species. / Master of Science