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In this thesis I use Drosophila melanogaster as a model organism to study the roles of reproductive proteins in determining male and female fitness. Many of these proteins are likely involved in mediating sexual conflict between the sexes, therefore I focus on how males and females interact at the molecular level, in order to gain insight into the mechanisms underlying sexually antagonistic coevolution. I provide the context for the work (Chapter 1) and describe the general methods and stocks used throughout (Chapter 2). I show that the sex peptide receptor (SPR) found in females, dramatically changes the fitness benefits to males after early rematings (Chapter 3), I also describe my investigation into the structure of the mating plug formed within females mated to males lacking the mating plug protein, PEBII (Chapter 4). I then test two candidate genes, Acp26Aa and Spn2, for roles in sperm competition and compare the results obtained from functional tests and correlative studies (Chapter 5). Next, I focus on the requirement of sex peptide (SP) for SPR and vice versa for inducing feeding responses in mated females and early changes in post mating egg laying and receptivity (Chapter 6). Carrying on from this, I investigate the role of SP and its related protein, Dup99B, in eliciting post mating responses in females (Chapter 7), Finally, I summarise the findings from my thesis and discuss ideas for future work to increase our understanding of the consequences of sexual conflict and sexually antagonistic coevolution in Drosophila melanogaster (Chapter 8). My research shows that reproductive proteins play important roles in determining male and female fitness and provides further data supporting how sperm competition and molecular interactions between the sexes can generate and maintain genetic variation for sexual traits.

Meiotic recombination in humans is essential for the faithful segregation of chromosomes during meiosis and is key in generating genetic diversity. Inferring past events from contemporary SNP haplotypes and studying de novo events in sperm DNA has shown that recombination occurs within 1-2 kb wide ‘hot spots’ in the human genome. In terms of male recombination, the pseudoautosomal regions (PARs) are especially interesting. PAR1 undergoes obligatory crossover in male meiosis and therefore constitutes a male-specific recombination ‘hot’ domain (rate approximately 20-fold above the genome average). It is thus ideally suited to sperm DNA studies. PAR2 is not essential in male meiosis, but nonetheless recombines at a rate >6-times the genome average. Despite this, relatively little is known about the fine-scale distribution of recombination in either pseudoautosomal region. To address this, linkage disequilibrium analysis and high-resolution sperm typing was used in this work, in order to identify and characterise a collection of PAR hot spots. This survey led to the identification of five active PAR hot spots, thereby providing relatively easy access to crossovers and noncrossovers. A second hot spot was identified in the SHOX region, providing information on hot spot spacing in PAR1 and facilitating important comparisons with autosomes. The first PAR1 double hot spot, a potential resource for investigating crossover interference, was also identified. Data from the two PAR2 hot spots (SPRY3 and PAR2A) provided direct evidence of hot spot activation by trans-acting PRDM9, with different protein variants activating either hot spot. The strongest meiotic drive observed at any human hot spot was also identified at SPRY3, providing insights into likely mechanisms of hot spot evolution. Finally, an extensive survey at SPRY3 provided unprecedented insights into the relative frequencies of crossovers and noncrossovers at hot spots and highlighted the presence of a second pathway of noncrossover-formation in humans.

Cohesin mediates sister chromatid cohesion (SCC), and its regulated association with chromatin is required to promote faithful chromosome segregation during mitosis and meiosis, as well as for the efficient repair of DNA double strand breaks (DSBs). In the mitotic cell cycle loading of cohesin requires a conserved complex containing the Scc2INipbl protein, which has also been proposed to promote binding of the cohesin-related complexes condensin and SMC-5/6. However, little is known about the factors that promote loading of cohesin and related SMC (structural maintenance of chromosomes) complexes during meiosis. During a screen for meiotic mutants in C. elegans, I isolated an allele of sec- 2, scc-2 (jql), that has allowed me to determine the roles that SCC-2 plays during meiosis. I show that during C. elegans meiosis loading of cohesin, but not condensin 11 or SMC-5/6, requires SCC-2, demonstrating that loading of condensin 11 and SMC-5/6 can be achieved by mechanisms independent of both SCC-2 and cohesin. The lack of cohesin in scc-2 mutants impairs the repair of meiotic DSBs and recombination intermediates accumulate extensively. Surprisingly, these accumulated intermediates fail to induce an apoptotic response, which is the normal outcome when persistent DNA lesions are detected by the conserved pachytene DNA damage checkpoint. I observed that this defect is caused by a failure to load the DNA damage sensor 9-1 ~ I complex onto persistent recombination intermediates in scc-2 mutants. A lack of meiotic cohesin also impairs the timely loading of the RAD-51 recombinase to irradiation-induced DSBs. These findings suggest that meiotic cohesin is required in the early steps of DSB processing and for the recruitment of checkpoint proteins to sites of DNA damage, thus revealing novel roles for cohesin.

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Meiotic recombination in Saccharomyces cerevisiae is initiated by the formation of DNA double strand breaks (DSBs), which are created by Spoil protein. Recombination preferentially occurs between homologous chromosomes, in order to establish interhomologue connections. These connections serve as a platform for genetic recombination and to promote accurate homologue disjunction at the first meiotic division (MI). Specific mechanisms are in place to ensure that meiotic DSB repair is directed towards interchromosomal repair, and genes thought to be involved in these mechanisms were examined in a DSB assay, where interchromosomal repair was precluded. Genes involved in the formation and processing of Spol1-DSBs were also examined. In meiosis, the regulation of resectioning is critical to repair outcome, and this assay was designed to measure two different lengths of resection tract. In a mek1 mutant, there was an increase in the generation of longer resection tracts, suggesting that Mek1 protein may exert its influence over repair template choice by negatively regulating DSB resectioning. An sae2 mutant was found to generate fewer shorter resection tracts, and was delayed for DSB repair. This suggested that Sae2 protein may have an early role in resectioning, by influencing repair template choice. Mutants of the MRX complex were all compromised for DSB repair, while an exol mutant failed to generate long resection tracts only. Finally, from work on a dmcl mutant, the prospect of protein sequestration at sites of excess single stranded DNA was proposed.