Analysis of Caenorhabditis elegans Sperm Number, Size, Activation, and Mitochondrial Content

Infertility is a widespread and often unexplained issue. Studying reproduction using C. elegans males offers insight into the influence of individual factors on male fertility in humans. We have created a collection of protocols to assess several aspects of C. elegans sperm quality, including number, size, rate of activation, and mitochondrial morphology. Studying sperm biology in a model system such as C. elegans allows access to the wealth of resources and techniques that have been optimized for that organism while providing valuable biological information that may be applicable to other systems. Graphic abstract: Flowchart depicting the preparation of C. elegans males and subsequent sperm quality assays


Data analysis software
We use GraphPad prism, although any software capable of t-test will suffice. ANOVA may be helpful if comparing multiple conditions but not strictly necessary.

Procedure
A. Prepare slides (to be used in D. Sperm size assay; E. Sperm activation assay; F. Qualitative mitochondrial morphology assay; and G. Quantitative mitochondrial fusion assay).
1. Using a PAP pen, draw circles of 1-1.5 cm in diameter on the microscope slides.
Note: One standard slide will easily fit two grease circles, with room to cover each individually (when using 22 × 22 mm square cover slips).
11. Mount samples on microscope slides (regular slides, not those prepared in Procedure A) with Vectashield mounting medium.
12. Cover with a cover slip and seal the edges with nail polish. Allow to dry (in the dark) for 5-10 min.
13. Image at 40-63× with DIC and DAPI filters, ensuring all spermatids are visible in each worm.

Note: Spermatids should be restricted to the seminal vesicle in male worms and can be distinguished from other germ cells by their compact nuclei.
14. Collect z-stacks of each seminal vesicle, ensuring all spermatids are captured (use 40× if needed to zoom out to include all spermatids). A representative image is shown in Figure 1.    Note: Activation assays are done using SM buffer containing BSA rather than Dextrose. We have tested with both supplements and noted that BSA is needed for activation. Note: We have found that sealing the slides with nail polish affects activation and thus use only petroleum jelly to seal slides for this assay. 10. After 15 min, cover the dissected animals with the prepared cover slip. Do not seal further. 11. Image with a 100× objective using a DIC filter. A representative image is shown in Figure 4.  c. Score at least 100 spermatids per replicate.  5. Transfer 5 cleaned virgin males to the SM buffer on the slide using either an eyelash brush or a platinum pick, depending on how animals were cleaned (see Step D1a). 6. Under a dissecting microscope, dissect worms to release the sperm (see Procedure D for instructions).
7. After dissecting the final male, add 25 μl of SM buffer containing BSA and JC-1.
a. Be careful to avoid completely removing the liquid from dissected spermatids, as this will affect spermatid physiology (and therefore mitochondrial phenotypes).
b. Be careful to avoid overflowing the grease circle and losing the sample. If necessary, use smaller wash volumes (80-90 μl).
10. Cover the dissected animals with a cover slip and seal with nail polish.
Representative images are shown in Figure 6.
Note: JC-1 is a mitochondria-specific dye that reflects changes in membrane potential. The ratio of red fluorescence (high membrane potential mitochondria species) to green fluorescence (low membrane potential mitochondria species) is a commonly used metric of mitochondrial health (Sivandzade et al., 2019).
a. Collect z-stacks of spermatids to be scored.

Count spermatid nuclei in z-stacks.
Note: To avoid counting the same nuclei more than once or losing track of the ones counted, divide the seminal vesicle into multiple sections by drawing lines and counting one section at a time. For reference, in a 40× stack of images for one seminal vesicle, we selected 50-80 sections.
The number of sections will depend on how the worm lays and how much mounting medium is between the cover slip and the slide (the smaller the volume, the flatter the worms may be, and fewer z-stacks will be needed to acquire the image, whereas more counting sections may be needed).
3. Each replicate should consist of at least 10 animals. 4. At least three separate replicates should be completed. Statistical analysis should be performed using the Student's t-test.