Worm Breeder's Gazette 4(1): 38
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
When males are dissected in M9 buffer the sperm that are released are invariably spherical cells whose diameter is ~4 . They have a prominent nucleus and often project long filamentous processes. When dissected in a low ionic strength HEPES buffer supplemented with BSA, 5-15% of the cells are not round, but rather extend a single pseudopod, 1-3 cell diameters long. These pseudopods change their shape and carry numerous small projections which appear to continuously move back towards the cell body, in a manner similar to ruffles on leukocytes, e.g. [See Figure 1] Pseudopods may attach to substrate, and well-attached cells translocate in the direction of the pseudopod. The cells migrate at about 10 /min (compared to 100 /min for amoebae or l /min for fibroblasts), and may have intermittent periods of quiescence, or of 'ruffling' and shape change without translocation. We refer to the pseudopod as the anterior part and to the cell body as the posterior part of the sperm. Two representative tracks are shown below. Time is in minutes. [See Figure 2] The production of pseudopods is dependent on the low ionic strength of the medium. TRIS can replace HEPES and 10mg/ml PVP can replace the 1mg/ml protein in the medium. The most important variable in pseudopod production is the mating activity of the males. In virgin males the fraction of pseudopod- bearing sperm is always low, but increases with age, except in UNC males, where the number of activated sperm is always low. In mated males the fraction of pseudopod-bearing sperm increases up to 80% with time of mating (it plateaus after 2 hrs). We routinely get ~50%. Hermaphrodites contribute little towards activating the sperm, as males mated to heat-killed hermaphrodites show the same level of sperm activation as those mated to live animals. The genotype of the mated hermaphrodites has no effect. Our working hypothesis is that, as in Ascaris, the male gonad ( probably the vas deferens) produces a sperm activating substance which may be released when the male engages in mating activity or in auto- erotic or homosexual activity. This activity is eliminated in paralyzed males. Transmission EM of pseudopod-bearing sperm reveals a striking asymmetry in the distribution of organelles. The nucleus, mitochondria and M.O.'s (=membranous organelles) are all contained within the posterior part of the cell, while the pseudopod contains no obvious substructure (in our present preparation procedure). We have examined most of our fer mutants by light microscopy with timelapse recording and by scanning electron microscopy on parallel samples. fer-1 sperm can be stimulated to make abortive pseudopods: they are very short but retain a 'ruffling' surface. These pseudopods are barely visible in the light microscope. The level of stimulation is the same at permissive temperature (in which the pseudopods have a normal shape) or restrictive temperature, and approximates wild-type stimulation. Two alleles of this gene (hc1 and hc24) have essentially identical morphology. However, some HC24 sperm are less stunted than HC1 sperm, which correlates with the greater leakiness of this allele. fer-2 sperm are asymmetric - they are capable of extending one or a bundle of long spikes that are sometimes branched. Other cells show elongation of the cell to a shape reminiscent of an incomplete pseudopod which is devoid of the 'ruffles'. fer-3 sperm are indistinguishable from those of wild-type. fer-4 sperm can form pseudopods, but the cells are immotile and the pseudopod membrane does not 'ruffle'. SEM micrographs reveal elongated pseudopods with ragged profiles and smooth surfaces. In addition, fer-4 sperm are variable in fer-5 and fer-6 sperm do not form structures which resemble normal pseudopods. Many cells of each strain exhibit large cavities and collapsed membranes. A few sperm have large spikes, some of which have protrusions reminiscent of pseudopod membranes, suggesting that these spikes are abnormal pseudopods. In summary, we have mutant strains each with a distinct sperm morphology which can be arranged in order of increasingly defective phenotype: fer-3<fer-1< -2<fer-5--fer-6. All but the last two extend pseudopodial structures and the fraction of cells bearing these structures is increased at least five-fold by mating. Even fer-5 and fer-6 sperm have the potential to become asymmetrical.