Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.
Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.
The molecular mechanisms by which spermatozoa (referred to as sperm) navigate through the female reproductive tract toward the oocyte are not well understood, yet are fundamental to sexual reproduction. Sperm motility is highly dynamic and depends on robust communication signals that alter sperm velocity and directional motility1,2,3,4,5,6,7,8,9,10,11,12. C. elegans has become a powerful model for studying sperm movement in vivo because the hermaphrodite’s transparent epidermis permits the tracking of live sperm at single cell resolution2,3,8,10. The purpose of this paper is to provide methods for assessing sperm movement within the C. elegans hermaphrodite uterus.
In animal species where sperm and oocyte meet in the external environment (i.e., acquatic environments), sperm respond to chemotactic signals secreted by oocytes. These signals guide the direction of sperm movement, bringing them closer to the signal source4,6,11. However, much less is known about sperm movement in species that fertilize internally. A major challenge is the architecture of the female reproductive tract, which is inaccessible to microscopy in most species. In vitro studies in humans, mice, and pigs, for example, provide evidence that subpopulations of sperm can respond to chemoattractants, fluid flow, and thermal gradients1,5,7,9,12. With these systems, the inability to visualize and track sperm movement in vivo places serious limitations on strategies to discover the key mechanisms regulating these functions.
To overcome these limitations, we have developed methods using the nematode C. elegans to directly visualize sperm after insemination, to measure individual sperm migration parameters in vivo, and to measure the ability of a sperm population to target the fertilization site. These methods, together with the C. elegans molecular and genetic toolset, facilitate discovery of the chemical signaling molecules and molecular machinery that regulate sperm motility behaviors. For instance, genetic screens can be conducted in hermaphrodites or males to identify genes that are essential for efficient sperm movement in vivo13. Molecules can be injected into the hermaphrodite gonad to test for effects on sperm activation, migration velocity, and directional motility3. Additionally, the described methods can be used to monitor rogue sperm migration into ectopic body locations and to evaluate sperm competition10,14.
C. elegans exist in nature as hermaphrodites and males (see Figure 1). The hermaphrodite gonad has two U-shaped arms that are mirror images of each other. During the L4 larval stage, the most proximal germ cells (i.e., the cells near the spermatheca) undergo spermatogenesis. Each primary spermatocyte enters meiosis and produces four haploid spermatids. These spermatids are pushed into the spermatheca along with the first mature oocyte and undergo spermiogenesis15. Adult hermaphrodites switch from spermatogenesis to oogenesis. The oocytes mature in an assembly line fashion along the gonad, with the most mature oocyte at the proximal end of the gonad, next to the spermatheca. MSP signals from the sperm is needed to trigger meiotic maturation and ovulation16,17. Male C. elegans, on the other hand, have a J-shaped gonad that produce only sperm. The spermatids are stored in the seminal vesicle. Upon mating with the hermaphrodite or female, the male inserts the spicules near the tail into the vulva. Spermatids are activated during ejaculation, when they come in contact with the seminal fluid18. C. elegans sperm do not swim as they are not flagellated. Instead, they crawl through the reproductive tract, using the pseudopod for locomotion. It is well established that male sperm, which are larger in size, have a competitive advantage over hermaphrodite sperm14.
In this method, male C. elegans act as the sperm donor and are mated to adult hermaprhodites. Adult males are stained with a fluorescent mitochondrial dye to produced labeled sperm. Once deposited through the hermaphrodite vulva, the sperm must crawl around the embryos in the uterus towards the spermatheca, or fertilization site. The transparent epidermis of the C. elegans model allows for the direct visualization of each individual sperm as it navigates through the female reproductive tract. In recent years, our lab has successfully used this method to demonstrate the importance of a class of F-series prostaglandins in guiding sperm from the vulva to the spermatheca19,20. The molecular mechansims governing its synthesis by the hermaphrodite and response by the sperm are still under investigation. However, this method for assessing sperm motility and migration greatly facilitates the identification of the key players that control sperm and oocyte communication in internally fertilizing animals. The following protocol describes step by step how to perform this assay.
NOTE: All steps in this protocol are performed at room temperature (~20-22 °C) or in constant temperature incubators set to 16 °C or 20 °C. Male and hermaphrodite C. elegans are grown using standard culture conditions and NA22 or OP50 E. coli as a food source21,22. Wild-type N2 hermaphrodites and fog-2(q71) males are used in the procedure below.
1. Day 1: Picking L4 Stage Hermaphrodites for Mating
2. Day 1: Staining of Males with Fluorescent Mitochondrial Dye (Mito-dye)
3. Day 2: Mating
4. Day 2: Mounting Worms for Visualization
5. Day 2: Image Acquisition Setup
NOTE: Any upright miscroscope equipped with epi-fluorescence, 10x and 60x objectives, and a digital camera can be used to acquire images for sperm distribution. Software capable of acquiring time-lapsed images are required for assessing sperm speed, directional velocity, and reversal frequency.
6. Quantification
To generate the results depicted in this paper, fog-2(q71) males were stained with the mito-dye and mated to wild-type, N2 hermaphrodites. Figure 2 provides an overall scheme for the method, including worm preparation, mating, and analysis. As Movie 1 shows, the adult hermaphrodite reproductive tract has two arms that are mirror images of each other. Upon mating, labeled sperm are deposited in the hermaphrodite uterus through the vulva. The sperm move around the developing embryos within the uterus toward the spermatheca, where they are stored until fertilization. As germ cells in the adult hermaphrodite develop into oocytes, the proximal, most mature oocyte is pushed into the spermatheca via sheath cell contrations. Fertilization occurs while the oocyte is in the spermatheca.
To quantify sperm distribution and migration through the female reproductive tract, the hermaphrodite uterus is divided into three zones (Movie 1, Figure 3A). Zone 1 spans the first third of the uterus, starting from the vulva. Zone 2 spans the middle third of the uterus, and Zone 3 spans the last third of the uterus and includes the spermatheca. Proper sperm guidance using wild-type, N2 hermaphrodites and fog-2(q71) males should result in approximately 90% of the labeled sperm reaching the spermatheca, or Zone 3 (Figure 3B). Matings that result in too few (Figure 3C, less than 10-15 sperm) or too many (Figure 3D, uterus filled with sperm) sperm in the uterus should not be counted. In matings that result in less than 10-15 sperm, 3-4 rogue sperm may heavily skew the data. Similarly, when the uterus is filled completely with sperm, the sperm cannot migrate appropriately. Sperm may seem scattered throughout the uteri of some mutants that display poor sperm guidance phenotype. However, in this case, sperm should not fill each crevice of the uterus, as seen in Figure 3D. Quantification of each arm of the gonad is considered one sample, or one n.
Time-lapse images are taken to quantify sperm velocity and reversal frequency. Only sperm in Zone 2 should be tracked (Figure 4A) because sperm in Zones 1 and 3 (Movie 1) tend to move in a circular pattern even within wild-type animals. Time-laspe images taken at 15-30 s intervals are usually used to track sperm. Only sperm that can be followed in consecutive frames for more than 2.5-3 min are quantified. In Figure 4B-M, the sperm marked by the red and blue dots satisfy this criterion, while the sperm marked by the green dot does not. Therefore, the values defined in Figure 4N are quantified for the sperm marked by the red and blue dots (Figure 4O), while those for the sperm marked by the green dot were not quantified.
Figure 1: Cartoon of the adult C. elegans hermaphrodite and male. Major reproductive structures are labeled in the figure. Please click here to view a larger version of this figure.
Figure 2: Schematic diagram of sample preparation and data acquisition. Males stained with the mito-dye are mated to synchronized adult hermaphrodites. Time-lapse images of mated hermaphrodites are taken immediately after mating to capture data for sperm velocity and reversal frequency. Still images of mated hermaphrodites are taken 1 h after mating to assess sperm distribution within the uterus. Refer to the text for more details. Please click here to view a larger version of this figure.
Figure 3: Quantifying sperm distribution within the hermaphrodite uterus. (A). Schematic of the C. elegans hermaphrodite uterus. V = vulva, E = embryo, S = spermatheca, O = oocyte, Z1-Z3 = Zones 1-3 used to measure sperm distribution. (B-D). DIC+TRITC merged (left panels) and TRITC only (right panels) images of the wild-type hermaphrodite uteri 1 h after mating to fog-2(q71) males stained with the mito-dye. Sperm appear red. Yellow outlines indicate the location of the spermatheca. Scale bar: 20 μm. Z1, Z2, Z3 quantification in B represent the percent sperm in each zone ± standard deviation. Images in C and D represent matings that have resulted in too few (C) or too many (D) sperm for quantification. Please click here to view a larger version of this figure.
Figure 4: Quantifying sperm velocity and reversal frequency during migration through the uterus. (A). DIC+TRITC merged image of a hermaphrodite uterus containing fluorescent sperm (red). V = vulva, yellow = spermatheca, Z1-Z3 = three zones of the uterus, black box: zone 2. (B-M). Time-lapse TRITC channel images zoomed in on zone 2 (black box in A). Images were acquired at 20 s intervals. 3 individual sperm were tracked in each image (red, green, and blue dots). Colored dots in panel M represents the path of each sperm from B-L. Scale bar = 20μm. (N). Equations and definitions for sperm speed, vector velocity and reversal frequency. (O). Speed, vectorial velocity and reversal frequency of sperm tracked in panels B-L by the red and blue dots. Please click here to view a larger version of this figure.
Movie 1: Movie of sperm movement and migration. A wildtype hermaphrodite was mated to fog-2(q71) males stained with mito-dye. The movie is a composite of time-lapse images taken at varied time intervals. Please click here to view this video. (Right-click to download.)
The ability of sperm to navigate the convoluted female reproductive tract and find oocytes is critical for sexual reproduction. Recent studies using sperm of internally fertilizing animals suggest they actively respond to various environmental cues, including chemical signals, fluid flow, and temperature gradients1,5,7,9,12. However, these observations have largely resulted from in vitro experiments and little is known about sperm behavior and communication within the reproductive tract. One of the main barriers to acquiring in vivo data on sperm migration and motility is the lack of visibility within most female reproductive tracts. The method we have described here using C. elegans overcomes this limitation. As the representative results demonstrate, the transparent epidermis allows for direct visualization and tracking of each sperm at single cell resolution in a live, intact organism.
C. elegans has two sexes. The males, with an XO genotype, produce only sperm, and in this method, are used as the sperm donors. The hermaphrodite, with an XX genotype, are modified females. Their gonads first undergo spermatogenesis during the fourth larval stage and switch to oogenesis in adulthood25. This assay uses adult hermaphrodites, whose reproductive tissues provide a model for the female reproductive tract. The utilization of both sexes in this assay allows us to identify genetic and molecular pathways in both the male and female that may regulate sperm guidance and motility. Combined with the whole host of genetic and molecular techniques available for C. elegans, this method can lead to novel insights into sperm migration and motility as well as sperm and oocyte communication.
A few critical steps in this protocol warrant further consideration, in addition to the details provided in the protocol section.
Worms
fog-2(q71), him-5(e1490), or him-8(e1489) mutant males can be used in place of N2 males. These mutations increase the frequency of males in cultures, but do not affect male mating or sperm functions13. Females, such as fog-2(q71) females, may be used in place of hermaphrodites. However, females must be pre-mated with males to allow proper oocyte development. The presence of fertilized embryos from this pre-mating also ensures that the uterus is long enough to properly assess sperm distribution. If mutant or experimental hermaphrodites are being assessed, include a control group(s). For example, mutant hermaphrodites should be paired with wildtype N2 hermaphrodites as a control for other variables in the assay. Hermaphrodites that have been fed with bacteria containing plasmids for RNA interference assays should also be fed with bacteria containing empty vector control. The distance from the vulva, where sperm are inseminated, to the spermatheca, the fertilization site, can vary depending on the number of eggs in the uterus (i.e., the uterus expands with increasing egg number). If comparisons between genotypes are made, select hermaphrodites whose uteri contain similar numbers of eggs. Do not select hermaphrodites containing hatching embryos or moving larvae. A time course may be performed to identify the optimal age at which hermaphrodites should be assayed.
Picking hermaphrodites and males
It is important that the hermaphrodites picked for this assay are not from overgrown or starved plates. Food and pheromone cues modulate the expression of DAF-7, a TGFß homolog. The DAF-7 pathway has been shown regulate the synthesis of F-series prostaglandins that play important roles in guiding sperm to the spermatheca20. Picking hermaphrodites from overcrowded or starved plates may result in poor sperm guidance not related to the target of interest. The density of the plates do not seem to affect the male sperm. However, males that are too young or too old may result in decreased mating efficiency (i.e., the percent of hermaphrodites on the mating plate that have enough sperm in their uteri to quantify). 1-3 day old adult males are optimal for this assay23.
Anesthetizing hermaphrodites
In our hands, the Tetramisole and Tricaine combination ensures that worms are immobilized, and remain alive during mating and image acquisition. Other anesthetics, such as sodium azide, may also be used. However, sodium azide is highly toxic and conditions need to be standardized. Immobilization techniques using microbeads, agarose, and microfluidics chambers are not recommended as they interfere with mating.
Male staining
The mito-dye used in this manuscript, MitoTrackerCMXRos, has been widely used for labeling sperm, as well as other mitochondria, in C. elegans. The sperm labeled with this mito-dye is fully functional, retaining its ability to be activated, migrate, fertilize oocytes, and produce viable progeny26,27. Other mitochondrial dyes, such as rhodamine 6G and DiOC6 have been used to stain C. elegans mitochondria28,29. However, conditions for these dyes need to be standardized for labeling sperm in this assay. In addition to mitochrondrial labeling mechanisms, DNA stains, such as Syto17, may also be used to label sperm for migration assays30. While these labeling techniques are relatively easy and quick to perform, transgenic strategies may also be employed to generate sperm that express fluorescent tags under sperm specific promoters31,32.
Mating
Mating dots that are too thick may decrease mating efficiency. Care should be taken to transfer as little bacteria as possible when transferring males and anesthetized hermaphrodites onto the mating dot.
Making agarose pads and placing the cover slip
Air bubbles can be generated in the agarose pads. They may refract light during image acquisition or, when large enough, cause worms to fall through, making it impossible to acquire the image(s). Similarly, air bubbles may be created along the hermaphrodites when the cover slip is placed over them on the agarose pad. These bubbles refract light and lead to decreased image quality. Practice will help decrease the occurrence of air bubbles.
Quantification
When quantifying sperm distribution, different z-planes through the uterus of a worm may have slight differences in sperm distribution. We find that taking a single image focused on the spermatheca gives us reproducible results that are similar to results obtained by averaging multiple z-sections. We recommend focusing the image on the center of the spermatheca, but the focal planes can be altered slightly based on the experimenter’s needs. It is critical, however, that all images are taken in the same manner. Furthermore, it is important that only sperm that are in focus are counted. It is the counter’s discretion in determining the criteria for in-focus sperm. However, it is critical that the criteria is applied systematically to every worm that is quantified. For sperm tracking, many software offer automatic tracking capabilities. However, we find that manual tracking outperforms the software’s automatic tracking algorithm for two main reasons: 1) The abundance of similarly sized nuclei within the confined space makes it difficult for the software to distinguish between individual sperm and create defined ROIs for each sperm. 2) As sperm go in and out of focus, their intensities shift, making it difficult for the software to keep track of the sperm over extended periods of time.
The authors have nothing to disclose.
We sincerely thank our late mentor, Dr. Michael Miller, for his inspiring and selfless mentorship and creation of this method as a tool to better understand sperm and oocyte communication. His sudden passing has been a tremendous loss for his family, his lab, and the scientific community. This study was supported by the NIH (R01GM085105 to MAM and F30HD094446 to MH). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Reagents and Material | |||
60mm x 15mm Petri Dish | Fisher | FB0875713A | |
Agar | Fisher | BP1423-500 | |
Sodium Chloride | Fisher | S671-3 | |
Peptone | Fisher | BP1420-500 | |
Cholesterol | Sigma-Aldrich | C8667 | |
LB broth, Miller | Fisher | 1426-2 | |
Escherichia coli strain NA22 | Caenorhabditis Genetics Center (CGC) | NA22 | Either this or OP50 E. coli can be used for C. elegans maintenance and assay. Both may be purchased at the CGC |
N2 | CGC | N2 | |
fog-2(q71) | CGC | CB4108 | |
Platinum wire 0.25mm dia | Alfa Aesar | 10288 | |
5 3/4" Disposable Pasteur pipet | Fisher | 13-678-20A | |
Watch glass | Fisher | 02-612A | |
5mm Dia. Glass rod | Fisher | 50-121-5269 | |
MitoTracker CMXRos (Mito-dye) | Fisher | M7512 | Shield from light, store at -20°C |
Monopostassium phosphate | Fisher | P285-500 | |
Disodium phosphate | Fisher | S374-1 | |
Magnesium sulfate | Fisher | M63-500 | |
Dimethyl sulfoxide | Fisher | BP231-1 | DMSO |
Aluminum foil | Fisher | 01-213-102 | |
Ethyl 3-aminobenzoate methanesulfonate | Sigma | E10521-10G | Tricaine is the common name. Store in aliquotes at -20°C. |
Tetramisole hydrochloride | Sigma | L9756-5G | Store in aliquotes at -20°C |
Agarose | Fisher | BP1356-100 | |
Coverslips | Fisher | 12-548-A | 18 x 18-1 |
Frosted microscope slides | Fisher | 12-552-3 | |
Name | Company | Catalog Number | Comments |
Equipment | |||
16°C and 20°C incubators | Fisher | 97-990E | Same model, set at different temperatures. |
Upright Microscope with epi-fluorescence illuminator, camera, and 10x and 60x objectives | Nikon | ||
Software with image acquisition and tracking capabilities | Nikon | NIS-elements AR | |
Stereo-microscope | Nikon | SMZ800N | Any stereo-microscope that can be used to visualize C. elegans may be used with this protocol |