Blog Post 2: Motility Experiments and Results

In my last post, I mentioned the process by which nematode spermatids activate to form spermatozoa. Sperm activation in nematodes is characterized, among other changes, by the formation of a pseudopod. Whereas vertebrate sperm “swim” using a flagellum, C. elegans sperm “crawl” using a pseudopod. The goal of both is to locomote towards and ultimately fertilize an oocyte.  In C. elegans, the process by which male sperm activate is tightly regulated because successful fertilization requires sperm to activate at precisely the right moment – when the male sperm are transferred to the reproductive tract of the hermaphrodite (Stanfield and Villenueve, 2006). If sperm activate precociously (within the male), then they are often unable to be transferred to a hermaphrodite.  Many proteins control this process of sperm activation to ensure that spermatids activate properly. One such protein is SWM-1, a protein that functions outside of sperm to inhibit sperm activation in males prior to insemination of the hermaphrodites. In swm-1 mutants, however, sperm activate precociously and often fail to be transferred to a hermaphrodite. This precocious activation phenotype extends to other mutants as well, including spe-6 mutants. In spe-6(hc187), the sperm of males raised at 25°C lack SPE-6 protein and activate precociously within the seminal vesicle.

If the spe-6 protein functions in wildtype sperm to inhibit precocious sperm activation, we wondered if it might also affect the motility of activated, crawling spermatozoa. To examine this question, we decided to examine the motility of spe-6 mutant sperm through video analysis.

In order to carry out these motility studies, swm-1 and spe-6(hc187) mutant males were raised at 25°C and isolated beginning at the L4 larval stage (the last larval stage before adulthood) to ensure celibacy. The one millimeter long worms were “dissected” by placing them in a drop of buffer on a slide and placing a coverslip on top of the slide and gently pressing down until the gonad was extruded from the worm’s body. The slides were then examined using DIC microscopy at 600X magnification. Once spermatozoa were identified, time-lapse videos were taken (one picture was taken every four seconds) and I used the video frames to track the speed of moving spermatozoa. The average velocity for each sperm was determined by selecting three five-frame tracks where a spermatozoon was moving at a constant rate. The average velocity was calculated for each of these tracks and then the mean velocity for the three tracks was calculated. Fifteen to seventeen moving sperm from each mutant were analyzed. The hc187 sperm had an overall average velocity of 0.1219 μm/s, while the swm-1 sperm had an average velocity of 0.1274 μm/s. Below is a bar graph comparing the overall average velocities for each worm type.

Average Velocity of Sperm

The standard deviation, used as error bars in the graph above, for the average velocity of all the hc187 sperm was 0.0625, while the standard deviation for the average velocity of all the swm-1 sperm was 0.0527. Combined with the mean of each worm type, this information was used to conduct an unpaired t-test between the two mutants. The p-value yielded was 0.793, well above the significance level of 0.05. That would suggest that there is no difference in the motility of spe-6(hc187) or swm-1 sperm, and therefore SPE-6 may not play a role in the velocity of sperm motility. However, the n value for this experiment was extremely low – less than twenty sperm from each mutant were studied, so this experiment could be improved by increasing the number of sperm studied.

In my last few days in lab I hope to finish up the last of the data analysis from the motility studies and prepare strains of worms for the fall. I hope to investigate the role SPE-6 plays in sperm after they have activated because many proteins necessary for sperm activation disappear after spermatids mature into spermatozoa, but SPE-6 remains in activated spermatozoa, which could indicate that it has other functions beyond inhibiting sperm activation.

* You may have noticed that I switched between uppercase notation for proteins (e.g. SWM-1) and lowercase italics (e.g. swm-1). Uppercase is used to identify a particular protein, while lowercase italics are used to denote the gene responsible for a particular protein product.

Works Cited

Stanfield, Gillian M., and Anne M. Villenueve. “Regulation of Sperm Activation by SWM-1 Is Required for Reproductive Success of C. elegans Males.” Current Biology 16.3 (2006): 253-62. ScienceDirect. Elsevier. Web. 12 June 2015. <http://www.sciencedirect.com/science/article/pii/S0960982206010244>.