A FUNCTIONAL, COMPARATIVE AND CLINICAL ANALYSIS OF SPERM-BORNE OOCYTE ACTIVATING FACTOR, PAWP

Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization.

Contact zone dynamics and the evolution of reproductive isolation in a North American treefrog, the spring peeper (Pseudacris crucifer)

Despite over seven decades of speciation research and 25 years of phylogeographic studies, a comprehensive understanding of mechanisms that generate biological species remains elusive. In temperate zones, the pervasiveness of range fragmentation and subsequent range expansions suggests that secondary contact between diverging lineages may be important in the evolution of species. Thus, such contact zones provide compelling opportunities to investigate evolutionary processes, particularly the roles of geographical isolation in initiating, and indirect selection against hybrids in completing (reinforcement), the evolution of reproductive isolation and speciation. The spring peeper (Pseudacris crucifer) has six well-supported mitochondrial lineages many of which are now in secondary contact. Here I investigate the evolutionary consequences of secondary contact of two such lineages (Eastern and Interior) in Southwestern Ontario using genetic, morphological, acoustical, experimental, and behavioural evidence to show accentuated divergence of the mate recognition system in sympatry. Mitochondrial and microsatellite data distinguish these two lineages but also show ongoing hybridization. Bayesian assignment tests and cline analysis imply asymmetrical introgression of Eastern lineage nuclear markers into Interior populations. Male calls are divergent between Eastern and Interior allopatric populations and show asymmetrical reproductive character displacement in sympatry. Female preference of pure lineage individuals is also exaggerated in sympatry, with hybrids showing intermediate traits and preference. I suggest that these patterns are most consistent with secondary reinforcement. I assessed levels of post-zygotic isolation between the Eastern and Interior lineages using a laboratory hybridization experiment. Hybrid tadpoles showed equal to or greater fitness than their pure lineage counterparts, but this may be countered through competition. More deformities and developmental anomalies in hybrid tadpoles further suggest post-zygotic isolation. Despite evidence for pre-mating isolation between the two lineages, isolation appears incomplete (i.e. hybridization is ongoing). I hypothesize that potentially less attractive hybrids may circumvent female choice by adopting satellite behaviour. Although mating tactics are related to body size, genetic status may play a role. I show that pure Eastern males almost always engage in calling, while hybrids adopt a satellite tactic. An absence of assortative mating, despite evidence of female preference, suggests successful satellite interception possibly facilitating introgression.

Do protected areas mitigate the effects of fisheries-induced evolution on parental care behaviour of a recreationally-important teleost fish?

Human-induced selection on animals and plants has been highly influential throughout our history and resulted in both intentional benefits and unintended detriments. Fisheries-induced evolution (FIE) describes the unintended selection on wild fish populations by fishing that has resulted in the evolution of exploited populations. While the use of aquatic protected areas that exclude angling might be considered an evolutionarily-enlightened management approach to dealing with issues arising from FIE little is known about the effectiveness of this approach for maintaining the phenotypic diversity of traits in protected areas versus those outside of their boundaries. In species that exhibit parental care, including the largemouth bass (Micropterus salmoides), active nest guarding and aggression towards potential brood predators by males increases the survival of offspring. This aggression may render these individuals particularly vulnerable to capture via angling as a result of increased propensity to attack fishing lures near their nests. Relative levels of aggression by these males during the parental care period correlate with their vulnerability to angling year round. Inasmuch as this parental behavior is heritable, this selective removal of more aggressive individuals by anglers should drive population-average phenotypes towards lower levels of aggression. To assess the effectiveness of protected areas at mitigating FIE, I compared the nest guarding behaviours of wild, free-swimming male bass during the early nesting period for bass within and outside protected areas. I found that nesting males within long-standing fishing sanctuaries (>70 yrs) were more aggressive towards captive bluegill sunfish (Lepomis macrochirus) placed directly on their nests, and patrolled larger areas around their nests compared to bass outside of sanctuaries. Males within protected areas were more likely to strike at artificial fishing lures and more prone to capture during experimental angling events. Collectively, my findings suggest that recreational angling selects for individual bass with lower levels of parental care and aggression, and that the establishment of protected areas may mitigate potential FIE. The extent to which this phenomenon occurs in other species and systems likely depends on the reproductive strategies of the fishes being considered, their spatial ecology relative to sanctuary boundaries, and habitat quality within protected areas.