Because of the small size of sperm and the technical challenges of observing events taking place inside the complex reproductive tract of females, there are numerous, fundamental aspects of reproduction that remain poorly understood in internally fertilizing species, including humans. This project is a detailed investigation of genetic, physiological and behavioral aspects of events taking place between insemination and fertilization in a model system: the fruit fly Drosophila melanogaster and closely related species. For the first time, these events will be directly observed in real time using unique transgenic fly strains producing sperm with heads that express green (GFP) or red fluorescent protein (RFP). Experiments will explore mechanisms of sperm migration, sperm storage, egg fertilization, the mechanisms by which sperm from different males compete to fertilize the eggs of twice-mated females, and mechanisms of ejaculate-female incompatibility contributing to reproductive failure in hybrid matings between closely related species.
The proposed research has the potential to generate watershed advances in the fields of reproductive physiology and genetics, sexual selection and speciation. Resulting progress in our understanding of sperm behavior within females and the identification of candidate genes contributing to sperm-female incompatibility are further likely to lead to advances in our understanding and treatment of human infertility.
Knowledge of events occurring between insemination and fertilization is critical to our understanding of (i) basic reproductive biology in internally fertilizing species, (ii) the causes of reproductive incompatibility for many infertile male-female pairs (both humans and threatened/endangered species subject to assisted reproductive technologies), (iii) and the selective causes of biodiversity. Because females of most species mate with multiple males, sexual selection continues after mating in the form of "sperm competition" to fertilize eggs and "cryptic female choice" among alternative males’ sperm or ejaculates. Such postcopulatory sexual selection is now recognized as one of the most potent forces driving diversification. We now recognize that sperm, seminal fluid biochemistry and female reproductive tracts (morphology, physiology and biochemistry) are some of the most rapidly evolving traits known. Moreover, although our knowledge is severely limited at present, ejaculates and female reproductive tracts are recognized as interacting in complex ways. Indeed, numerous lines of evidence suggest that interacting sperm and ejaculate traits coevolve with female reproductive tract traits – a consequence of postcopulatory sexual selection (including sexual conflict). Finally, because ejaculate-female compatibility are central to successful reproduction, whenever populations are geographically isolated from one another, any rapid and independent coevolutionary changes in interacting ejaculate-female traits may result in reproductive incompatibility between members of the different populations should they later have the opportunity to interbreed. This sequence of events might be a widespread cause of new species formation. Testing such hypotheses and furthering our knowledge of such basic reproductive biology has been impeded, however, due to two experimental challenges. First, the small size of sperm and the technical challenges of observing events taking place inside the complex reproductive tract of females have hampered progress. Second, it has not been possible to discriminate between the sperm of different males within the female tract. The research supported by this grant solved these challenges through the genetic engineering of unique, transgenic strains of Drosophila melanogaster and related species (model study organisms) that produce sperm with heads that express green fluorescent protein (GFP) or red fluorescent protein (RFP), respectively. This material enabled, for the first time, unambiguous discrimination among sperm from different males within twice-mated females, as well as direct observation of real-time interactions between sperm from different males in vivo. Using this unique research material, we successfully completed all of the proposed research, which included (i) determining the processes and sperm-female interactions underlying variation in male competitive fertilization success, (ii) examining the extent to which interacting male and female traits, as well as the processes by which they interact, have evolutionarily diverged across three closely related species: D. melanogaster, D. simulans and D. mauritiana, and (iii) determined the mechanisms of ejaculate-female incompatibility underlying reproductive isolation in hybrid matings between D. simulans and D. mauritiana. Our results have confirmed that ejaculate-female interaction processes are indeed evolving strikingly fast, and that such diversification can directly lead to speciation, or at least maintain genetic barriers between closely related, hybridizing species. Our unique research material further revealed that sperm exhibit a striking degree of mobility and complex behavior within the female reproductive tract. This discover has generated a novel research program on sperm behavior in vivo. With regard to broader impacts of this research, Our fluorescent sperm stocks have been requested by laboratories worldwide pursuing diverse research programs investigating various aspects of reproduction, already leading to several publications (Köttgen et al. 2011; Sartain et al. 2011; Schnakenberg et al. 2011). Educational contributions include undergraduate learning modules developed using the transgenic lines at Cornell University and Gustavus Adolphus College, and use of movies and images associated with a forthcoming textbook. Wide coverage of Manier et al. (2010, Science), included documentary and public venues (e.g., NSF’s Science Nation; Pitnick volunteered as scientific consultant on the evolution of sex and sexuality for the Discovery Network’s series "Curiosity"). The PI and Co-PI’s laboratories involve undergraduates actively recruited from the McNair Scholars Program and Collegiate Science and Technology Entry Program, which includes socioeconomically underrepresented students. This NSF grant supported 9 undergraduate research experiences, with 6 of these meriting co-authorship. The PI and all Co-PIs have also begun working with the Milton J. Rubenstein Museum of Science & Technology (MOST) to provide mentoring, public seminars, and research facilities for students interested in reproductive biology. Students from the Syracuse City School District, with a large proportion of minority and economically disadvantaged students, will be particularly encouraged to participate.
