Blog 4: End of Year UGR Report

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.

 

 

 

 

 

 

Blog Post 3

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.