Worm Breeder's Gazette 9(3): 98

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Genetic and Phenotypic Analysis of Spermatogenesis-Defective Mutations in C. elegans

S. L'Hernault, D. Shakes and S. Ward

Figure 1

Figure 2

We have been analyzing mutations that affect speratogenisis in C. 
these were isolated in our laboratory by 
selecting sterile mutants that produced large number 9 of oocytes that 
could be fertilized by wild type male sperm; others were sent to us 
from other labs. All mutations were assigned to linkage groups and 
tested for complementation to all other known linked spermatogenesis-
defective mutations. Many of the genes identified have been mapped. At 
this time, there are 50 mutations which we have organized into 32 
complementation groups; seven complementation groups have more than 
one allele. For mutations in 9/10 genes tested, the phenotype over a 
deletion is identical to the homozygous mutant phenotype 80 the sperm 
defect seems to be the null phenotype.
Most of our recent work on this project has been limited to linkage 
group I where we have identified 28 mutations in 12-13 complementation 
groups. We have recovered many of these mutants as cis doubles with 
dpy-5. This has facilitated the linkage assignment, balancing of 
nonconditional mutants and identification of sterile worms for 
phenotypic analysis (see below). Depending on their position on the 
map, nonconditional steriles have been balanced to sDp2 or a 
complementing LG I deficiency. Five linkage group r complementation 
groups contain  ore than one allele. Our present understanding of the 
genetic organization of these LG I spermatogenesis-defective mutants 
appears in Figure 1.
We have analyzed the light microscopic phenotypes of most of our LG 
I spermatogenesis-defective mutants and many on the other five linkage 
groups (see summary Table and Fig. 2). The top row of cartoons in Fig. 
2 shows the pathway of normal spermatogenesis. Just below this pathway 
fer and spe genes that arrest development at an apparently normal step 
in the pathway are shown at the step they arrest. Aberrant phenotypes 
that accumulate in some mutants are shown below the pathway. For 
example, spe-4 mutations accumulate cells that look like spermatocytes 
but have 4 haploid nuclei; fer-2, 
s accumulate aberrant looking 
spermatids which fail to activate to spermatozoa. Mutations in five 
genes (fer-7, cumulate normal looking 
spermatozoa that are motile in vitro, but these fail to fertilize 
oocyt of them are swept out of the hermaphrodite spermatheca by the 
passage of oocytes, but others are retained.
Previously, we thought sperm-defective sterile hermaphrodites that 
laid large numbers of oocytes were likely to be defective in the 
postmeiotic stages of spermatogenesis where the spermatid is 
transformed into a motile spermatozoa. While this is frequently the 
case, we have also recovered mutants defective in the earlier stages 
of spermatogenesis (see Fig. 2).
We have decided to name all new sperm-defective genes spe, 
irrespective of their sperm-defective phenotypes. We will abandon the 
further use of the gene name fer which was intended to distinguish 
that subset of sperm-defective mutants that accumulate haploid gametes.
There are now a number of intermediate phenotypes that make this 
distinction less useful.
We are interested in cloning the genes that have been identified by 
our collection of spe and fer mutating. Unfortunately, it has not 
proven possible to isolate mutants out of 'high hopper' lines because 
there is a very high background of oocyte laying. For instance, we 
used Mike Finney's cleaned up 'high hopper' line (originally created 
in Phil Anderson's lab) in a precomplementation screen to fer-1(hclts),
dpy-5(e61)r; 15ts),unc-4 (e120)II and recovered 
oocyte layers at a frequency of 1 out of 30. We tried to recover a 
nonconditional sterile from 17 of these lines and were not successful. 
Since this did not look like a particularly promising approach, we 
have sought other methods. Fortunately, it appears that the spe-4 gene 
has already been cloned. Genetic analysis indicates that spe-4 
complements sDf5 but fails to complement sDf6. This places this gene 
very close to unc-15 which is in a contig identified by Allan Coulson 
and John Sulston. This same contig also contains a lambda phage that 
Dan Burke had identified as containing a sequence that hybridized 
specifically to male RNA in a differential hybridization assay. We are 
trying to determine the relationship (if any) of this lambda phage and 
the spe-4 gene; even if there is not a relationship it seems likely 
that this contig contains the spe-4 gene. We are also trying to clone 
the fer-15 gene, which seems to be blocked in the spermatid-
spermatozoon transition. This gene has been positioned near emb-27 on 
LG II; it complements mnDf 92 but fails to complement mnDf91. Phil 
Carter has been kind enough to send us his 100 kb cosmid walk from the 
mnDf 103 breakpoint towards fer-15. We hope to walk and jump through 
this region to clone the interval between the mnDf 91 and 92 
breakpoints that define the fer-15 gene. Differential hybridization 
assays and/or microinjection might then permit identification of the 
fer-15 gene.
{Figures 1,
2}

Figure 1

Figure 2