Research involves critical thinking, problem solving and patience. Researching about infertility has helped me realize how this issue has impacted so many people. The process of researching has enhanced my critical thinking skills. Any questions that came along the path of research required me to consult with the latest literature published in various scientific journals. I was able to practice problem solving and most of all I was able to learn from my mistakes. From the start of researching to now I have come a long way and I have greatly enhanced my research skills. I was also able to present my findings at the Society of Fellows event at Pace University and won an award for my research. I am truly grateful for the Undergraduate Research Grant which helped fund my project.
Mammalian fertilization begins with the fusion of two gametes, sperm, and egg. The spermatozoa enter the female reproductive tract and are required to migrate to the oviduct. The oviduct is where the spermatozoa meet the ovulated eggs. But the question is, how do these specialized cells know when and how to interact. At the cellular level, fertilization is a complex molecular interaction that may require multiprotein complexes on both gametes for sperm-egg fusion to occur. However, this process often is unsuccessful because of genetic mutations in the sperm. We have previously identified a sperm membrane protein interactome in Caenorhabditis elegans (C. elegans) that provided evidence that sperm membrane proteins interact extensively. We hypothesized that these interactions are dynamic during spermiogenesis and sperm maturation and are necessary for fertilization. In the interactome, FER-1 interacts directly with four other membrane proteins, including SPE-10. This project leverages the plethora of genetic resources available for C. elegans research to analyze how double mutant animals with mutations in fer-1 & spe-10 (VC40852), fer-1 &spe-9 (VC20575) , and fer-1 & spe-12 (VC20650) impact fertility.
One hypothesis to explain the decrease of hermaphrodite self progeny of the double mutants (VC20650 and VC40852) is that the fer-1, spe-12 and spe-10 are required for successful fertilization similar to spe-36. It is possible that VC20650 and VC40852 double mutants are also embryonic lethal. RNA interference experimentation is necessary to determine the location of lethality during fertilization. Due to unforeseen circumstances the semi-quantitative assays were not completed at 16°C and 20°C. By determining the rate of fertility of the double mutants are these two remaining temperatures we will have a better understanding of why the mutants are infertile. Furthermore, we did not expect to see an increase in fertility for the VC20575 strain. This is interesting because the double mutant had a significant increase in fertility not only compared to the other mutants but the control (N2) C. elegans.
Further experimentation at 16°C and 20°C is needed to determine if there is any decrease in fertilization. As hypothesized before, since single mutations significantly decrease the rate of fertility, it is reasonable to assume that double mutations will result in a more significant decrease in the rate of fertility. However, if a significant increase in the rate of fertility is shown, as in the VC20575 strain, it can be concluded that either the mutations do not impact the encoded protein or one of these mutations changes the molecular complex formation and could compensate for the loss of one of these functions. Various genetic approaches have provided insight into genes necessary for sperm viability — understanding how one gene influences another will offer a comprehensive understanding of the network design underpinning infertility.
Over the past few months, I have made progress in the lab and gained positive results. To evaluate the functions of specific genes required for male fertilization, I looked for articles in the literature. I came across an assay that is used by many scientists to determine the rate of fertility in the model organism C. elegans. I used the Brood Sizing Assay to determine the rate of three different strains at 25 degrees Celsius. This experiment requires me to monitor the rate of fertility of the C. elegans daily and to accurately count the number of progenies produced.
During this process, I came across multiple problems. The first problem that occurred was that one of the strains became contaminated. To resolve this issue, I kept transferring the worms to new plates until the contamination reduced. I then began the assay, and this required me to use 90 plates per week. To do this, I had to manage my time well and make plates quickly and efficiently. The first batch of plates I made was not the best since the bacteria overgrew. I made the plates again, and they came out perfect. I will be using the new plates this week to re-do the assay at 20 Degrees Celsius. So far, I have seen that at 25 degrees Celsius, the mutants had reduced fertility compared to the control group meaning these mutations result in fertility defects.
Fertilization is a complex molecular interaction that may require multiprotein complexes on both gametes for sperm-egg fusion to occur. My project aims to identify how potential multiprotein protein complexes are functioning and what their impact is on fertility and sperm-egg fusion. We have previously identified a sperm membrane protein interactome in Caenorhabditis elegans (C. elegans) that provided evidence that sperm membrane proteins interact extensively. My lab team has weekly meetings where we discuss our projects for the following week. I have been maintaining my worms and making sure they are healthy for future experiments. One of the difficulties I have faced is that one of my plates got infected, and this required me to follow better sterilization procedures.
We are analyzing the spe-36 gene, which was originally identified as a sterile strain. Further analysis revealed that spe-36 mutants are unable to fertilize eggs normally, but the cause is unknown. To better understand why spe-36 mutants are sterile, we are analyzing the mutants at different developmental stages. We are investigating three different strains N2 (wild-type), spe-36(as1), and spe-36(as1)asEx96. The spe-36(as1) mutant is the C. elegans without the gene of interest. And the spe-36(as1)asEx96 is the C. elegans with the gene of interest replaced and tagged with green fluorescent protein (GFP). We have conducted a brood size analysis at 16°C to assess fertility rate. We discovered that C.elegans with spe-36 mutants are unable to fertilize. When the gene is knocked in, the fertilization reaches close to the wild-type but not entirely. We are interested in seeing what will happen at 20°C and 25°C. We expect to see a slower fertilization rate at these temperatures since they are not the preferred temperature.
We have also identified two C. elegans strains with mutations in genes known to be necessary for fertilization to analyze the effect that missense mutations in multiple sperm membrane proteins have on sperm-egg fusion. Since I will be examining more than one mutation, we ordered different strains of C. elegans from the Caenorhabditis Genetics Center.The VC40852 strain has missense mutations in fer-1 and spe-10 and the VC20575 has missense mutations in fer-1 and spe-9. We are measuring the effect of these mutations on brood size and sperm-egg fusion. The analysis of both strains will provide insight into the genetic interactions between fer-1, spe-9, and spe-10 and allow us to generate a model of how these genes could be functioning together to mediate fertilization.
Male infertility can result from low sperm production, abnormal sperm function, or defects in sperm delivery. The mammalian fertilization process begins with the fusion of two germ cells. More specifically, the spermatozoa enter the female reproductive tract and are required to migrate to the oviduct. The oviduct is where the spermatozoa meet the ovulated eggs. However, this process often is not successful because of genetic mutations in the sperm. I am interested in determining how different genes mutated in sperm impact the rate of infertility.
More specifically, I am interested in analyzing how double mutations influence infertility. As a model to study sperm development, I am using the nematode Caenorhabditis elegans (C. elegans). C. elegans is a well-established genetic system that can be used to determine how my genes of interests control spermiogenesis and how their misregulation could lead to infertility. Using C. elegans as a model system, we are using a genome sequenced multi-mutation library, from the Million Mutation Project, to identify genes associated with defects in sperm. We will than analyze the rate of fertility of single mutants versus double mutants. We are going to use those orthologs and perform structure-function analysis using single mutants to understand better which domains of the gene the mutation impacts. In the end, this analysis will help us better understand infertility and will help us identify how specific genes interact with one another.
Dr. Deng and I have been working on Amber Tutorials and test running the program “Maestro Schrodinger.” In order for me to utilize the program Maestro, I need to be proficient in all of the tutorials on Amber. Amber is a tool that teaches people how to use a molecular dynamics software. The first step was to learn the Unix Command-Line. From this tutorial I learned how to use the command line to navigate a file system, view directories view directories and copy, move, and remove files and directories. Then I did another tutorial which gave an introduction to Molecular Dynamics Simulations. This tutorial was extremely helpful because it gave me a better insight into what we are actually researching. This research is extremely beneficial to me because I get to see how the latest technology can be used in science today.
The last two weeks we actually ran into a lot of problems. Once we received the license from Maestro to use the system we were not able to register the program. Therefore, we contacted the company and are waiting for a response. We also had issues trying to connect to the server. We worked with the IT department to resolve most of the issue. Currently, I am working on 2 more tutorials. Even though Amber is a great program the site does not work that often. These obstacles have taught me that in research most of the time things do not go smoothly. We need to work together to resolve unforeseen issues and not give up. Dr. Deng and I are excited to finally be able to use Maestro. We will be continuing our research this upcoming fall semester.
Dr. Deng and I are researching how drugs inhibit the HIV virus using molecular dynamic. This study will help and guide researchers in the drug discovery industry to better understand how such drugs work. This will help researchers design and create a better drug to treat HIV-AIDS virus. So far this summer I learned what molecular dynamics is and how it is used. I also learned how molecular dynamics became successful and what some of the challenges are. Molecular dynamics provides an atomistic insights about the structure and dynamics of proteins and nucleic acids.
The first Molecular dynamic simulation actually furthered our knowledge about proteins because it revealed the dynamic nature of a folded protein. I was able to apply my Biology 101 knowledge to the simulation once I saw the protein folding. Furthermore, I learned how a drug molecule finds its target binding site. Molecular dynamics actually simulates trajectories which provide an atomic level view of the binding process. This is essential in our research because we need to simulate a molecule that will perfectly fit into the binding site.
After reading multiple papers on molecular dynamics and HIV-AIDS we decided to use the program “Maestro Schrodinger” to dock the molecules. We ran into a few issues with the server at Pace and it took us a few day to solve the issue. Currently, we are using “Amber Tutorials” to learn the Unix Command-Line. Even though it’s only been a few weeks I have been able to apply a lot of my Biology 101 and 102 knowledge and I have learned a lot about the HIV virus and molecular dynamics. I look forward to continuing this research project.