A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.
Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm – around 300 – and are stored in a small ‘bag’ called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for >50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite’s tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of ‘Sperm Medium’. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.
1) Enrichment of male population
2) Identification and isolation of males
3) Dissection of males and in vitro activation
4) Visualization of spermtids and spermatozoa
5) Representative Results
An example of DIC image of spermatids isolated from male C. elegans is shown in Figure 1. Spermatids are spherical in shape and the nuclei are prominent. In vitro activated sperm are shown in Figure 2.
Figure 1. DIC image of C. elegans spermatids.
Figure 2. DIC image of in vitro activated C. elegans spermatozoa.
In addition to analyzing Spe mutants, this protocol has other important applications, such as analyzing sperm morphology with aging. Spermatids and spermatozoa isolated using this protocol can be used in other downstream experiments such as, immunostaining of wild type and mutant sperm8, 9, motility of sperm on slides10, physiological measurements9, 11, or even artificial insemination12.
The authors have nothing to disclose.
We thank Samuel Ward, Diane C. Shakes and Gregory Nelson for pioneering this technique. We thank Pavan Kadandale and Brian Geldziler for videos showing activated spermatozoa; members of the Singson lab for helpful discussions; CGC for strains and NIH for supporting us through grant (R01HD054681).
Material Name | Typ | Company | Catalogue Number | Comment |
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Tuberculin syringe | Becton Dickinson | 309623 | Syringe: 1 ml, needle: 27G X ½ in |
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Pap pen | Zymed | 00-8888 | ||
VWR VistaVision HistoBond Microscope Slides |
VWR International | 16004-406 | Dimension: 75X25X1mm |
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Cover glass | VWR International | 48366045 | ||
Protease | Sigma | P-6911 |