Investigating the Role of the Caenorhabditis elegans Gene M05D6.2, an Ortholog of Human T- Complex Protein (TCP11), in Sperm Function and Fertility
During the 2016-17 academic year, I have been conducting research with Dr. Matthew Marcello. Our research lab looks into different genes that could potentially affect fertility. Over the course of this undergraduate-faculty research program, I have continued my investigation on the gene M05D6.2 in C.elegans, whose ortholog in humans is known as Tcp11 (human t-complex protein 11). The Tcp11 gene is necessary for proper sperm morphology as well as fertility. This gene has been associated with the coiling of a sperms flagella and has been found to localize in both a sperm’s head and flagellum. The goal of our research is to establish the role that the C.elegans ortholog of Tcp11, M05D6.2, plays in regards to fertility and ultimately, determine the molecular role of the TCP11 gene domain, which is still unknown. Previously, we were able to conclude that the M05D6.2 gene is necessary for male reproduction in C. elegans. After conducting research for the past few months, we have not been able to add much new data.
I mentioned in my most recent blog post that obtaining males can be a very time consuming process. Since males naturally occur so rarely we are force them by inducing reproductive stress onto L4 worms with knocked out genes of interest. The way we induce this stress is to subject the worms to increased temperatures which is supposed to give an increased yield of male progeny in ~2 days.
Previous to me, undergraduate researchers that started the project were able to determine that M05D6.2 is essential for sperm activation and fertilization because hermaphrodites with the gene knocked down produced less progeny. After they visualized this, the worms underwent DAPI staining which is a fluorescent dye that stains DNA. This staining revealed that the worms were still able to produce the same amount of sperm although they were unable to reproduce at the same rate. Next various mutants of M05D6.2 were created with the entire gene knocked down, one to resemble the same single nucleotide polymorphism (SNP) that is seen in infertile male humans, and one with a GFP tag on the proteins N-terminus. The fertility of the worms was then analyzed and visualized under a microscope where nothing was seen due to weak fluorescence staining. We were able to travel to William Patterson University in Wayne New Jersey to present this work at a poster session. This was my first time presenting in a professional setting which I enjoyed very much. Usually I get very nervous when presenting but this time was much different from what I expected. The setting was professional yet also very casual and it was really exciting to be able to talk with other scientists who were very obviously interested in our work.
For future directions, hopefully next semester I will take over this project and I will repeat the analysis of the mutant hermaphrodite’s fertility since the data cannot be considered conclusive yet since it was only conducted once. Secondly, I will re-do the GFP staining to hopefully get a good visualization of where the protein localized within the worm. Then hopefully I will conduct the same experiments with male mutants to really get a good idea of how this can possibly be related to male infertility in humans and possible bring many couples happiness. It would be beneficial if I could continue this work over the summer but since I live out of state I will be forced to resume next semester and hopefully have better luck obtaining males.
Since my last post, things have been somewhat slow in the research lab. We are aiming to conduct experiments on male worms. To obtain male worms of the correct mutant strain. First, we have to conduct RNAi on wild-type worms. Once these worms have successfully grown into their L4 stage we pick them off and place them on a separate plate which we then place in an incubator which is ~5 degrees Celsius higher than their optimal growth temperature. This is called heat shocking. The reason we do this is that under increased temperatures, the worms experience reproductive stress which is supposed to give a higher yield of male worms, which at standard temperatures occur only 0.1% of the time by spontaneous non-disjunction. After the worms are in the intense heat for about 6 hours we then transfer them back to the standard temperature and await their reproductive cycles completion. After 2 days we then check the plates every few hours for 2-3 days in hopes of finding male worms. So far we have been unsuccessful and so we will continue to repeat these steps until we find males so that we can proceed with more experiments.
Since my first blog post, I have learned a lot of techniques commonly used in a genetics research laboratory. I have made plates on which the worms will grow, spotted them with bacteria for RNAi, and cultivated different mutant strains of worms and observed various phenotypes. To set up this experiment, the first thing I did was grow my bacterial cultures, which contained siRNA, that would be used to silence a targeted gene. The bacteria were then incubated for 16 hours. After 16 hours a 100 microliter spot of each bacterium was placed on a RNAi plate and incubated at room temperature for 24 hours.
The next morning I set up my experiment, placing 5 L4 N2 C.elegans hermaphrodites on each plate and incubating them at 22 degrees Celsius overnight. A week later I observe the phenotypes produced and repeat the experiment. Along with collecting the data from these experiments, I have also been learning how to perform statistical analysis using a program called R.
I will be continuing a project started by a previous undergraduate researcher which consists of investigating the role of certain genes in the fertilization of Caenorhabditis elegans necessary for sperm capacitation that can be compared to a similar complex in humans. The gene we will be focusing on, M05D6.2, is an ortholog for human t-complex protein 11 (TCP11) which is a testis specific gene product. TCP11 is hypothesized to be essential for proper sperm activation in mammals. Men with mutations in the M05D6.2 ortholog have fertility issues. By understanding the function of this gene, we could help these patients find better treatment or determine if they need to seek assisted reproductive technologies to have children.
Previously used techniques in the lab include RNAi interface which has revealed that males exposed to the treatment show a decrease in fertility while hermaphrodites exposed to the same treatment have no reduction in fertility. Using my background as a technician in the Biology department, a research assistant in a pathology lab at the University of Pennsylvania , and working with my supervisor I plan to present my work at the annual Eastern Colleges Science Conference in Spring 2018 and publish my results in a peer-reviewed journal when it is complete. My goals include being able to participate in undergraduate research until graduation from Pace where I then will apply to attend graduate school with a PhD track. Research will fit into these goals because it is my ultimate career goal when I finish schooling.