Spermatozoa are the most diverse cell type, exhibiting rapid and dramatic evolutionary changes in form. However, the genetics and adaptive significance of sperm form are poorly understood. Investigations are proposed (1) to examine the relationship between sperm length and motility/behavior within the female reproductive tract, and (2) to identify genes of major influence on sperm length. These studies will be conducted with populations of the fruit fly Drosophila melanogaster that have been experimentally evolved to have unusually short or long sperm, and several closely related species differing substantially in sperm length. All populations have been genetically engineered to produce sperm whose heads glow green or red under fluorescent light. This material will enable, in the first investigation, unambiguous discrimination among sperm from different males within twice-mated females, as well as direct observation in vivo of real-time sperm motility and sperm-sperm and sperm-female interactions. The second investigation will use breakthrough gene sequencing technology (i.e., restriction-site associated DNA mapping) to identify genes associated with sperm length variation.
The proposed research has the potential to generate watershed advances in the fields of reproductive physiology and genetics, sexual selection and speciation. Many undergraduate and graduate students will be trained in the course of this work. Resulting progress in our understanding of sperm behavior within females and the identification of candidate genes contributing to sperm form are also likely to lead to advances in our understanding and treatment of human infertility and in the further development of assisted reproductive technologies used in the preservation of threatened and endangered species.
Intellectual merit: Evolutionary biologists have long been fascinated by reproductive traits, because they tend to evolve rapidly, are often involved in the generation of new species, and can feature dramatic and bizarre morphologies. Spermatozoa are no exception, although they all generally function to fertilize eggs. It is commonly recognized that interactions with the female reproductive tract can drive evolution of sperm traits, but how sperm function within the female’s sperm storage structures remains poorly understood. Understanding the functional implications of sperm morphological variation as well as its genetic basis is fundamental for elucidating the evolutionary significance of the wide array of sperm morphologies found in nature (Fig. 1). This NSF award supported two Projects that (1) identified candidate genes of major influence on sperm length and (2) quantified structure-function relationships between sperm length and velocity/behavior within the female reproductive tract. We chose the fruit fly, Drosophila melanogaster, as our study animal, because it is an important model organism for genetics, and males have unusually long sperm, almost 2 mm in length. For Project 1, we used two lab populations of D. melanogaster that were artificially selected for especially long or short sperm (sperm selection lines) and inbred them to genetically homogenize them and maximize fixed genetic differences between populations. We then bred the lines together for six generations (Fig. 2), and measured sperm length in all F6 sons (Fig. 3). We sequenced the sons’ genomes and identified 503 genetic differences (single nucleotide polymorphisms or SNPs) associated with sperm length. Of these, 83% reside within 206 genes, 76% of which fall within protein-coding regions (exons). Among these candidate genes, 33 encode sperm proteome components critical to flagellum development, over half of which are active when sperm are elongating during spermatogenesis. A substantial number of the top candidates encode components of mature spermatozoa or are upregulated during sperm elongation, and several are development genes not yet characterized for their spermatogenic function. Female D. melanogaster store sperm for up to several weeks in specialized sperm storage organs, including the primary organ called the seminal receptacle (SR; Fig. 4). For Project 2, we examined velocity and behavior of long or short sperm within long or short SRs. We used the same sperm selection lines as in Project 1, as well as a complementary set of populations previously selected for long or short SRs (SR selection lines). The sperm selection lines were genetically transformed to have a green fluorescent protein (GFP) in sperm heads, allowing visualization of individual sperm within the female reproductive tract (Fig. 5). We also examined sperm velocity and behavior of three closely-related species (D. simulans, D. mauritiana and D. yakuba), all transformed to have GFP sperm heads and varying in sperm and SR lengths. These experiments allowed us to quantify sperm-SR structure-function relationships between species. We previously documented a mechanism of reproductive isolation between D. simulans and D. mauritiana involving differences in sperm length, and one objective of this study was to identify differences in sperm function within each of these species as well as in hybrid matings. These experiments have been completed, and data analysis is underway. Broader Impacts: Because so little is known about the functional significance of sperm length and the genetics of sperm morphology, our results are generating watershed advances in the fields of reproductive physiology, sexual selection, and speciation. The unique GFP- (and RFP-) tagged D. melanogaster sperm lines we engineered have become a valuable resource to over 40 laboratories around the world. Widespread coverage of our recent report in Science provided an opportunity for us to work with numerous parties worldwide to incorporate our movies into media for classroom, documentary and public venues, to inform the public about reproduction, evolution and scientific inquiry (e.g., NSF’s Science Nation: www.nsf.gov/news/special_reports/science_nation/fruitflies.jsp). The PI and Co-PIs also became involved in efforts to improve and enhance diversity in science education in a nearby underserved high school with a graduation rate currently under 50%. Co-PI Manier initiated a relationship between the School of Education and the Biology Department and organized participation by in weekly science tutoring of underrepresented students coordinated by the School of Education. She also developed and taught a two-week Summer Science Institute for these students at Syracuse University in 2012 (Fig. 6); planning is underway for the 2013 Institute. This institute provided hands-on laboratory experience for students and additional teaching tools for three biology teachers. Entry and exit surveys showed students’ confidence in their science ability increased, and they were more likely to favor a science career. When asked to draw a scientist, students were also more likely to draw a woman. Two of the three biology teachers gained additional research experience in the PI and co-PI’s laboratories developing three genetics modules (supported by an RET Supplement to this grant).
