Despite a century of intensive research, fertilization is one of the least understood fundamental biological processes. Chemical signaling between gametes through fluid-borne cues occurs in diverse taxa with highly divergent reproductive strategies and is thought to play a fundamental role in reproduction. Still, it is unclear how chemical communication between gametes occurs under natural conditions. A critical determinant of the effectiveness of chemical cues and their influence on the motility of male gametes is ambient fluid motion. Fluid motion may promote cell interactions by bringing gametes together or alternately may inhibit adhesion and binding, yet very little is known about the effects of flow on the motility and chemotaxis of male gametes.

Existing methods have limited ability to study the role of physics and chemistry in mediating gamete behavior and fertilization. It is very difficult to accurately control fluid motion and chemical cues at microscopic scales. In this project, state-of-the-art microfluidic approaches will enable unprecedented control over microenvironments naturally inhabited by gametes. This study will take a comprehensive approach and apply microfluidics to determine the roles played by physics and chemistry in gamete interactions. The proposed research, to be carried out under the guidance of PIs Jeff Riffell (U. Washington), Roman Stocker (MIT) and Richard Zimmer (UCLA), is thus structured around two principal aims: (i) determine the impact of chemical cues on male gamete motility and on fertilization success; (ii) establish the effects of fluid motion on the motility of male gametes and their response to chemical cues. The synergy and complementarity of expertise between the three PIs will enable an in-depth characterization of the biomechanics of male gamete swimming and of chemical communication between germ cells.

The comprehensive and interdisciplinary approach of this study will have broad and diverse impacts on science and society. A better understanding of male gamete chemotaxis will arise from the use of microfluidic technology and provide new knowledge on reproduction and conservation biology. At the same time, the advances fostered by this study in attaining control of fluid flow and chemical cues at the microscale will provide a broad methodological framework for diverse areas of biology. The intimate combination of physics, biology and chemistry in this study will provide ample training opportunities for students at high school, undergraduate and graduate levels, emphasizing under-represented groups in science, through (1) a collaboration with the Summer Institute for Life Science (SILS) at the University of Washington, a 4-week hands-on summer institute that provides grade 4-8 teachers with research experience; (2) the development of a 3-hour science experience to be offered through MIT's Edgerton Center Outreach Program, designed for high-school students to promote hands-on experience in science; (3) the creation of a new course module at UCLA and the involvement of 3-4 UCLA undergraduates in research, each quarter; these undergraduates will be drawn from underrepresented groups through the UCLA CARE (Center for Academic and Research Excellence) and the UC LEADS (Leadership Excellence through Advanced DegreeS) Programs; and (4) the training of graduate students and postdoctorates in cell biology and microfluidics. Together, these programs will foster outreach and science education at multiple educational levels. Broad dissemination of results in technical and popular literature, in the tradition of all three PIs, will complement this outreach plan.

Project Report

" Jeff Riffell, Roman Stocker, Richard Zimmer Despite a century of intensive research, fertilization is one of the least understood fundamental biological processes. Chemical signaling between sperm and egg through waterborne cues occurs in diverse taxa with highly divergent reproductive strategies, including external and internal fertilizers, suggesting that these chemical cues play a fundamental role in reproduction. Yet understanding of how chemical communication between gametes occurs under natural conditions has been elusive. A critical determinant of sperm chemotaxis and gamete interactions is ambient fluid motion – a ubiquitous process for all sexual organisms, including both plants and animals. Fluid motion may play a critical role in mediating these interactions by (1) controlling the distribution of the chemical attractant around the egg; and (2) affecting gamete interactions either by bringing sperm and eggs together or, alternately, decreasing interactions by overwhelming the ability of the sperm to bind to the egg. The effects of flow on gamete interactions, and how it controls sperm motility and chemotaxis, have largely been undescribed. In this project, we used state-of-the-art microfluidic approaches to allow unprecedented control over both the physical and chemical environment of sperm cells. In addition, we used a combination of high-speed imaging and advanced image analysis to track the waveform of the sperm cells to identify the mechanisms that permit chemotaxis, and calcium imaging to determine how physiological changes in the cell correlate with the chemotactic response. We focused on three broad research foci: (1) determine how fluid forces affect sperm motility; (2) identify the chemoattractant conditions (eg, gradient and/or chemical concentration) that promote sperm chemotaxis; and (3) determine the simultaneous effects of fluid flow and chemoattractant conditions on sperm chemosensory-mediated behavior. An important first step in examining the effects of fluid motion on sperm motility is to identify the dynamics of the flagellar waveform under different fluid regimes. The flagellum effectively ‘steers’ the cell and if deformation occurs due to fluid motion, this could have important consequences for motility. We combined high-speed video microscopy with custom image analysis routines to accurately measure the flagellar waveforms at 400-1000 frames/s. Results of this analysis revealed that there were striking similarities in sperm swimming kinematics between genetically dissimilar organisms, ranging from marine invertebrates to humans. Moreover, using the free-spawning marine invertebrate, Lytechinus pictus, we found that sperm cells were sensitive to a specific range of shears; at low shears (0.2/s) cells could effectively navigate in the microfluidic channel, but at high shears (6-20/s) the distribution of cells was controlled by the fluid flow. The rich behavior observed in this system points toward complex coupling of cell motility with flow that has important implications for sperm transport and fertility in a number of terrestrial and aquatic organisms. We also found that sperm chemosensory responses were variable. When pooled across multiple individuals of L. pictus, we found that, when stimulated with the chemoattractant, sperm deviated from the path of their prior circular trajectory through a sequence of turns interspersed with periods of straighter swimming, the ‘turn-and-run’ pattern. Turning episodes coincided with an increase in calcium-evoked responses and chemotactic "run" towards the increasing gradient. Although the chemoattractant elicited a strong response in the sperm population, when it was tested with individual males the response was variable. To further explore if these differences in chemotactic abilities between males correlated with differences in fertilization success, we conducted a preliminary experiment by combining calcium imaging and sperm chemotaxis assays in the microfluidic device in parallel with fertilization assays using the sperm from individual males. Results from these experiments suggest that there was a significant correlation between fertilization and sperm velocity, and between fertilization and sperm orientation to the gradient, between individual males. Experiments are ongoing to further examine the links between sperm behavior and fertilization. As part of this effort, interdisciplinary training in engineering, microfluidics, cell physiology, and sensory ecology were provided for participating undergraduate and graduate students, and postdoctorates. The established collaboration (MIT, UW, UCLA) was facilitated by convened meetings and exchanges between PIs, students and postdocs. Finally, research generated from this project was implemented as modules in the PIs’ courses at MIT, UW, and UCLA.

Agency
National Science Foundation (NSF)
Institute
Division of Biological Infrastructure (DBI)
Application #
1120200
Program Officer
Joyce Fernandes
Project Start
Project End
Budget Start
2011-09-01
Budget End
2014-08-31
Support Year
Fiscal Year
2011
Total Cost
$433,413
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139