Dissertations / Theses on the topic 'Spermatogenesis. Drosophila melanogaster'

Drosophila Lasp contains a LIM domain, two nebulin repeats, and a SH3 domain, and exhibits high homology with mammalian Lasp family proteins. Vertebrate Lasp localizes to focal adhesions and to the leading edge of migrating cells and binds filamentous actin. To investigate Drosophila Lasp in vivo, we generated a Lasp null mutant, named Laspl, and showed that Laspl is male sterile. We observed two major functions of Lasp during Drosophila spermatogenesis. First, in the stem cell niche, hub cells fail to localize to the apical end of Drosophila testis in Laspl mutant. Hub cell anchoring is dependent on cell adhesion between cells and extracellular matrix (ECM), which is mediated by integrins. Lasp genetically interacts with betaPS integrin showing complete hub cell mislocalization. This indicates that Lasp is involved in an integrin-dependent process. However, hub cell anchoring is not required for fertility or stem cell maintenance. Secondly, we observe that actin cones, a unique actin structure during spermatid individualization, are perturbed in Laspl. Our data for Lasp expression in actin cones and incomplete individualization indicate that Lasp may play a role in tethering actin to the plasma membrane.

Mestrado em Biotecnologia - Biotecnologia Industrial e Ambiental
Cilia/flagella are microtubule (MT)-based oraganelle emanating from the surface of many eukaryotic cells. They are involved in a variety of processes including cell motility, fluid flow, and sensing processes. The skeleton of cilium, called axoneme, is templated from the basal body, a modified mature centriole required for centrosome formation. Mutations in human genes disrupting the function or structure of these organelles cause several human disorders, including infertility, ciliopathies, and cancer. In Drosophila melanogaster, cilia are present in the neurons and in the sperm. Defects in these structures and/or in its functions lead to clear phenotypes, such as delayed development, uncoordination and sterility. CEP164, a centrosomal protein, localizes at the distal appendages of the mature centriole. Mutations in CEP164 gene, in humans, cause Nephronophthisis, a ciliopathy that causes organ degeneration. The aim of this work consists of studying the CEP164 role in ciliogenesis and cilia function in sensory neurons and sperm in D. melanogaster. For that, we used two strategies: one involving knock down of CEP164 in neurons and sperm using the Gal4/UAS system, and other the generation of new excition alleles of CEP164 to study the effect of CEP164 in the fruit fly. In D. melanogaster olfactory neurons, CEP164 localizes at the connceting cilia, a transition zone equivalent structure. We observed by RNAi knock down that CEP164 is important for adult fly proper coordination. The cilia structures were affected in those flies, however, the fertility of those males was not affected.
Os cílios/flagelos são organelos constituídos por microtúbulos que emanam da superfície da maioria das células eucariotas. Estes estão envolvidos numa variedade de processos incluindo motilidade celular, movimento de fluidos e mecanismos de perceção de estímulos externos. O esqueleto de um cílio, designado axonema, é determinado por um corpo basal, que consiste num centríolo maduro/mãe modificado necessário para a formação de centrossomas. Mutações em genes humanos que interfiram na função ou estrutura desses organelos causam doenças como infertilidade, ciliopatias e cancro. Em Drosophila melanogaster, os cílios estão presentes nos neurónios e no esperma. Defeitos nestas estruturas e/ou na sua função leva a fenótipos como desenvolvimento tardio, falta de coordenação e esterilidade. A CEP164, uma proteína centrossomal, localiza-se nos apêndices distais do centríolo maduro. Mutações no gene CEP164, em humanos, causam nefronoptise, uma ciliopatia que causa a degeneração dos órgãos. O objectivo deste trabalho consiste no estudo da função da proteína CEP164 na ciliogenese e no papel dos cílios nos neurónios sensoriais e no esperma em D. melanogaster. Para tal, foram utilizadas duas estratégias: uma que envolve a depleção da proteína CEP164 em neurónios e no esperma utilizando o sistema Gal4/UAS, e a outra consistiu na criação de novos alelos de excisão de CEP164 para estudar o efeito desta proteína na mosca da fruta. Nos neurónios olfativos de D. melanogaster, a CEP164 localiza-se na base dos cílios, numa estrutura equivalente á zona de transição dos axonemas. Observou-se através de depleção por RNAi que a CEP164 é importante para a coordenação apropriada na mosca. A estrutura dos cílios foi afectada nessas moscas, contudo a fertilidade dos machos não foi afectada.

Inactive mutants of the ped gene cause two phenotypes in Drosophila melanogaster: male sterility and the early translation of DHODH within spermatogenesis. Investigation of the PED amino acid sequence revealed an OTU domain and an ubiquitin interacting motif, suggesting that it is a member of the otubain sub-family of de-ubiqutinating enzymes. To test this, the putative active cysteine residue was mutated. Results show that this single cysteine residue is required for ped to confer male fertility. Purified wild type PED was also used to carry out in vitro deubiquitinating assays. These assays failed to show any ability for PED to cut ubiquitin chains of varying length or linkage type. Previously, a translational control element was identified in dhod mRNA which is required for its early translation phenotype in ped mutants. In an attempt to identify additional transcripts that have their translational timing affected by PED, the don juan-like 5′ UTR was inserted into a reporter gene and examined in a ped mutant background. No delay of this reporter gene was observed suggesting that don juan-like mRNA is not under the exact control pathway that dhod is.

Iron has a dualistic character. On the one hand it is essential for the life of most organisms, on the other hand it is involved in the generation of reactive oxygen species that are implicated in diseases and aging. During evolution efficient mechanisms for uptake, handling and storage of iron in a safe way have developed to keep the balance between iron availability and minimizing the hazards. In eukaryotes, mitochondria are the central organelle for “metabolizing” iron and consequently play an important role in cellular iron homeostasis. Mitoferrins are mitochondrial carrier proteins, which are involved in iron transport into mitochondria. In vertebrates two mitoferrins exist, one (mitoferrin1) of which is essential for heme synthesis during erythropoiesis, while the function of the other (mitoferrin2) is not well defined. In the fruit fly we found only one mitoferrin gene (dmfrn), which codes most likely for a functional homologueof vertebrate mitoferrin2. In Drosophila cell culture, dmfrn overexpression resulted in an overestimation of cell sensed iron levels. The signal responsible for this, is most likely a yet unidentified compound of ISC synthesis. In the cell culture system we also showed that iron chelation blocks the progression of the cell cycle in a reversible and therefore most likely controlled way. Study of different dmfrn mutants indicates a role of dmfrn during spermatogenesis and development to adulthood. As dmfrn deletion mutants are not lethal, it is likely that other lower affinity iron transporters exist. A similar conclusion has been drawn by others from the study of yeast mitoferrin homologuemutants. Rim2p/Mrs12p has recently been implicated in mitochondrial iron transport, and might be an alternative metal carrier. We identified a putative homologuein the fruit fly and found a possible link between mutants in this gene and iron. Our results emphasize the importance of the mitochondrial iron metabolism in cellular iron homeostasis. We also show for the first time, a direct connection between the mitochondrial iron metabolism and spermatogenesis. Mutants characterized and developed by us will help to study these processes in further detail and reveal the underlying mechanisms.

MicroRNAs (miRNAs) are small, non-coding RNAs that act as regulators of gene expression at the post-transcriptional level in plants and animals. In animal cells, miRNAs typically bind with imperfect complementation to sequences within the 3′ UTRs of mRNAs, thereby inhibiting the translation of the transcripts. MiRNAs affect a variety of developmental pathways, and some of them appear to play important roles in defining the differential gene expression within the mammalian testis and during spermatogenesis; their functions in insects, however, remain largely unexplored. In this study, I examined the expression of several putative testis-specific miRNAs in different tissues and developmental stages of the mosquito Aedes aegypti and the vinegar fly Drosophila melanogaster. MiRNAs -9, -34, -100, -124, and -219 were all expressed in the testes of the two insects, but some differences in their expression in other tissues were observed. One particular miRNA, miR-34, was examined more thoroughly, and was confirmed to target genes that either have functions in spermatogenesis or have a testis-specific expression pattern in the two dipteran insects. Inhibition of miR-34, using antisense oligonucleotides, and RNA interference-mediated knockdown of its target, aae/014067, in A. aegypti negatively impacted the fertility of the mosquito males. These results suggest that both aae/014067 and miR-34 are clearly associated with A. aegypti male fertility and the disruption of their normal expression could render mosquitoes sterile, which would help in the development of sterile male release programs, to reduce the risk of mosquito-borne disease without the use of broad-spectrum pesticides that kill many non-target species.