[
Worm Breeder's Gazette,
1975]
Since observing that two of Jim Lewis's alleles of chemotaxis defective gene 1 (che 2) were sterile at 25 due to sperm dysfunction, much of the effort in our laboratory has been to determine the reason for this sterility. In order to do this, it has been necessary to study normal spermatogenesis and fertilization. One question that we have asked is what fraction of sperm in a wild-type hermaphrodite normally fertilize eggs? The experiment to answer this question was simply to follow a synchronized population of wild-type hermaphrodites determining the number of sperm remaining in each animal by using the Feulgen stain and determining the number of zygotes produced by counting progeny and fertilized eggs. Several time points were taken to be sure that sperm were indeed only synthesized during L4 and early adult as observed by Hirsh, Oppenheim, and Klass (1976) and others. The results of this experiment are summarized in the table below. [See Figure 1] Table Legend: The population was initiated by collecting animals hatched during a one-hour period. The sperm per worm was determined in Feulgen stained whole mounts by counting the small, densely staining, hollow looking nuclei characteristic of stained sperm. The sperm decrease is decrease in sperm from the maximum number. Zygotes per worm is the sum of progeny laid plus fertile eggs in the worms when fixed. Zygotes/sperm measures the efficiency of sperm utilization; the error shown is twice the standard error of the mean propagated from the errors in measured numbers. Oocytes per worm is the sum of the unfertilized eggs laid and those found in the gonad when fixed. The total sperm and progeny yield in this experiment were slightly lower than the 280/worm that we normally observe. It is apparent from Table 1 that the efficiency of sperm utilization is nearly 1. That is, every sperm normally fertilized an egg. Not every oocyte is fertilized because the worm produces about 100 more oocytes than sperm. That sperm are not synthesized after early adulthood was confirmed by the complete absence of the densely staining 'meiotic precursor' cell nuclei that mark the maturation of sperm from meiotic pachytene to spermatids in hermaphrodites and males.
[
Worm Breeder's Gazette,
1977]
The process of fertilization requires the interaction of gametes that must have specialized macromolecules on their surface that promote cell adhesion and penetration. By studying fertilization in detail and by isolating mutants blocked in fertilization, our laboratory hopes to investigate the genetic control of the cell surface architecture of the gametes. We have begun by studying the normal process of fertilization in C. elegans and the morphology of the sperm with light and electron microscopy. In the hermaphrodite, sperm accumulate in the spermatheca. They first contact an oocyte in the oviduct as the oocyte matures adjacent to the spermatheca. When the oocyte is mature, contractions of the oviduct wall push into the spermatheca where it contacts many sperm. It is then squeezed into the uterus through a constriction at the end of the spermatheca. Supernumerary sperm carried into the uterus on the oocyte abruptly migrate back through the constriction to the spermatheca so that every sperm can fertilize an oocyte. When males mate with hermaphrodites they deposit their sperm in the region of the vulva and these sperm migrate past the zygotes in the uterus to the spermatheca. When the male sperm arrive at the spermatheca they preferentially fertilize the subsequent oocytes even though hermaphrodite sperm are still present. The sperm are ameboid cells with a specialized pseudopodial region that is extended from the cell while the sperm is migrating. The sperm contain specialized membraneous vesicles that can fuse with the plasma membrane. In one fertilization defective mutant this fusion does not occur, in another the vesicles have altered fine structure. Sperm extracted into buffer from males or hermaphrodites initially appear as irregular round cells 5-7 m in diameter, but after a few minutes they begin to extend long filamentous processes that can grow to as long as 40 m. Such processes are not seen on sperm inside the hermaphrodite and their origin and role in fertilization is unclear.
[
Worm Breeder's Gazette,
1976]
We are continuing to study sperm morphology and the process of fertilization in order to characterize temperature sensitive fertilization defective mutants. We have begun studying the process of male fertilization. When wild-type males are mated to young wild- type adult hermaphrodites, the male sperm are deposited in the region of the vulva amidst the hermaphrodite zygotes. Within five hours all the male sperm migrate to the region of the spermatheca. Most surprisingly, five hours after mating some hermaphrodites will begin producing only outcross progeny. Thus not only can male sperm reach the spermatheca rapidly, but when they do so they fertilize oocytes preferentially in spite of the presence of hermaphrodite sperm. We are investigating the basis of both the male sperm motility and the mechanism of preferential fertilization. Sam will get back to studying chemotaxis yet. The temperature sensitive sterility of Che 1 mutants, E1034 and E1035, is not due to deficient sperm production. At 25 C spermatogenesis is normal and the mutant sperm have normal morphology by light and scanning electron microscopy. The sperm contact the oocytes in the hermaphrodite but fertilization does not take place. The mutant sperm are swept into the uterus and expelled when oocytes are laid. We are pursuing the nature of this defect by further scanning and transmission electron microscopy and by immunological and biochemical study of sperm membrane proteins. Several other temperature sensitive sterile mutants in different genes also make sperm that fail to function. We have not been able to resolve the question of whether the sterile defect in these strains is due to the same genetic defect as the behavioral and anatomical defects. Neither the behavioral nor the anatomical defects are temperature sensitive; we have been unable to revert the sterile phenotype and we have been unable to separate the phenotypes by recombination. Other alleles of the behavioral locus isolated in our lab and obtained from Dave Dusenbery are not sterile at 25 C. Perhaps we have a small deletion that alters two linked genes. Does anyone have direct evidence for EMS inducing deletions in C. elegans or any other organism? Does anyone know of a deletion that gives a temperature sensitive phenotype?