Dissezione genetica della spermatogenesi in Drosophila melanogaster: caratterizzazione genetica, citologica e molecolare di un gruppo di mutanti maschio-sterili coinvolti nel ciclo cellulare meiotico

摘要

My research was focused on a set of ethyl methanesulfonate (EMS)-induced male sterile recessive mutations mapping to chromosomes 2 and 3 in Drosophila melanogaster. The fruitfly is anamenable system to study spermatogenesis process for several reasons: ease of cytological analysis;availability of mutations at any step; and highly improved methods of genetic and molecular investigation. My study consisted in the genetic, molecular and cytological dissection of two groups of Drosophila male sterile mutants: twine-noncomplementing and twine-complementing mutants.Twine is a gene mapping on the left arm of chromosome 2 and encoding for a phosphataseresponsible for the triggering of the Cdc2-CyclinB complex, underlying meiotic entry. tweHB5 loss of function mutation causes the primary spermatocytes to fail both meiotic divisions, yet allowingsome further spermatid differentiation. However, the ensuing phenotype is sterile, since the sperms are not mobile. The set of EMS-induced male sterile mutations I’ve been handling showed a mutant phenotype resembling that of tweHB5, thus, implying the possibility that the twinenoncomplementing mutations were allelic to twine. Mapping of such mutations by meiotic recombination together with sequencing of twine region in the genome of these flies suggested that12-228, 60-40 and 54-23 mutations had two mutations on chromosome 2 selected for male sterility: one accounting for male sterility and the other one on twine gene, underlying the meiotic phenotype. The just mentioned results were also supported by the “cleaning” of the chromosome 2,carried out in these mutants by meiotic recombination. Different were the outcomes from 50-38 stock, another twine-noncomplementing mutation. In this case, recombination mapping and twine sequencing as well as chromosome “cleaning” suggested the presence of a single mutation on chromosome 2 of these mutants, close to twine gene, underlying both male sterility and meiotic phenotype. A detailed cytological analysis of this mutant by antibodies against some basic effectors of meiotic cell cycle, like α-tubulin to detect meiotic spindle, lamin for nuclear envelope and Spd2 for centrosomes, proved the failure of nuclear envelope breakdown and spindle assembly besides defects in number and localization of centrosomes in primary spermatocytes. Such anomalies underlie the failure of meiosis in 50-38 male mutants. Cytological characterization also revealeddefects during the later stages of spermatogenesis, consisting into mislocalization of basal body that prevented a proper cyst polarization.Among the twine-complementing mutations, one, named 71-43, resulted extremely interestingbecause of the following features. Mapping data by recombination and deficiency, and chromosome “cleaning” placed it on the distal portion of the right arm of the chromosome 2 as the only mutationpresent on this chromosome, responsible for meiotic and sterile phenotype. Cytological dissection of the spermatogenesis process in these mutants by means of antibodies provided the evidence of a panoply of anomalies, starting from young primary spermatocytes until to spermatids. In particular, defects of nuclear envelope, centrosomes and meiotic spindle were detected by immunofluorescence experiments in the early stages of male germ line development as well asimpairment of nucleus-basal body docking, cyst polarization and spermatid individualization processes in the later stages. The failure of assembly of a canonical meiotic cell cycle machinery accounted for the execution of only one, as well defective, meiotic division in 71-43 male mutants.71-43 homozygous mutant females showed reduced fertility, thus indicating that the function performed by this gene in oogenesis is likely redundant. A lower mitotic index in mutant larval brains in comparison with wildtype, pointed out that 71-43 acts also in mitosis, even though itsaction should be dispensable since no delayed development nor lethality are apparent. Towards the molecular identification of the gene mutated in 71-43 stock, different approaches were followed, both via forward and reverse genetics. Complementation analysis of males heterozygous for 71-43 mutation and a mutated allele of most of the genes embedded into the region where 71-43 was identified to lie, allowed to rule out all the genes so far tested as candidates for 71-43 locus.Proteomics approach, consisted in a comparative study of protein profiles of 71-43 homozygote vs 71-43 heterozygote testes, underlined the presence of some proteins misregulated in 71-43 mutants, whose genes however map outside the 71-43 genetic interval. Such an analysis, however, provided intriguing clues about the putative pathways where 71-43 may act and, therefore, new roads to pursue.