LaMunyon CW et al. (1995) International C. elegans Meeting "SPERM COMPETITION IN HERMAPHRODITIC CAENORHABDITIS SPECIES"

Ward S, LaMunyon CW

[International C. elegans Meeting, 1995]

As in C. elegans, male sperm take precedence over hermaphrodite sperm after C. briggsae worms mate. However, in C. briggsae the initial outcross progeny are principally hermaphrodites and become male-biased with time. Thus, X-bearing male sperm have a striking fertilization advantage over non-X-bearing male sperm. Results from controlled matings show that the X-bearing sperm neither outnumber nor activate faster than their non-X-bearing counterparts. The advantage (meiotic drive) of the X-bearing sperm is therefore due to a competitive edge over non-X-bearing sperm. Thus, in C. briggsae, male X-bearing sperm outcompete male non-X-bearing sperm which themselves outcompete hermaphrodite sperm. While we do not know the mechanism underlying the competitive effect of the X chromosome, we do have evidence from artificial insemination that the precedence of male sperm over hermaphrodite sperm in C. elegans is independent of both seminal fluid and the mode of sperm activation (male or hermaphrodite). Male sperm precedence in hermaphroditic Caenorhabditis maximizes outcrossing after mating, and the X-bearing sperm advantage in C. briggsae delays the associated cost of making males which are by themselves incapable of reproduction.

LaMunyon CW et al. (1997) International C. elegans Meeting "SIZE AND X CHROMOSOMES: DETERMINANTS OF SPERM COMPETITIVENESS IN HERMAPHRODITIC CAENORHABDITIS"

Ward S, LaMunyon CW

[International C. elegans Meeting, 1997]

Male sperm take precedence over hermaphrodite sperm in hermaphroditic Caenorhabditis. For C. elegans, we present evidence from mating and artificial insemination experiments that the male sperm advantage is neither numerical nor an effect of seminal fluid; it is, however, a consequence of sperm size. We found that male sperm are larger, crawl faster, and displace the smaller hermaphrodite sperm from the spermatheca. These results support comparative studies indicating that in at least some species, larger sperm are better competitors. Sperm competition in C. briggsae is similar to that in C. elegans, except that the outcross progeny show a biased sex ratio. Initially, the bias is toward hermaphrodites, the products of X-bearing sperm. Later outcross progeny are composed in large part of males, which result from nullo-X sperm. Thus, in C. briggsae, X-bearing male sperm have an unusual fertilization advantage over nullo-X male sperm. This X-bearing sperm superiority preserves the benefits of outcrossing while delaying an important cost associated with mating: the production of male offspring, which are incapable of reproducing by themselves. Thus, cell size and X chromosome content are determinants of sperm competitiveness which represent evolutionary strategies for maximized outcrossing with minimized cost.

Sokolich, T.J. et al. (2017) International Worm Meeting "Mutation of a highly conserved, intronic sequence prompts temperature-dependent aberrant splicing of precursor mRNA."

Shakes, D., LaMunyon, C.W., Sokolich, T.J., Prajapati, G.

[International Worm Meeting, 2017]

Traditionally, the study of essential and pleiotropic genes have posed unique challenges in the domain of reverse genetics. Knockout of essential genes can result in death or sterility, thus complicating phenotypic analysis. The complete inactivation of pleiotropic genes permits superficial investigation but may shroud the significance of independent expression events. Easily toggled between states, temperature sensitive mutations can abate some of these obstacles in gene function studies. The spe-6 gene of C. elegans is required for spermatid activation, major sperm protein assembly, and the proper execution of meiosis I. Two intronic mutations in spe-6 (zq18,and hc190) independently and in a temperature-dependent manner reduce the of function of this gene. At 25oC, a majority of spe-6(zq18) precursor mRNA is aberrantly spliced, subsequently invoking a reduction of fertility. At 15oC, fewer spe-6(zq18) transcripts are incorrectly spliced, and affected worms maintain normal fertility. spe-6(hc190) follows this same pattern of conditional fecundity. However, aberrant splicing of this allele has not been detected, possibly due to nonsense-mediated mRNA decay. The zq18 and hc190 alleles are characterized by guanine-to-adenine substitutions on the 5th and 1st bases, respectively, immediately flanking the 3' end of the exon. Furthermore, the 5' intronic sequence encompassing these two mutations is highly conserved amongst Eukaryotic genes and implicated in exon-intron junction recognition. Here, we report the effects of zq18- and hc190-like point mutations in other genes without a native restrictive temperature. MosSCI and the CRISPR/Cas9 system were used to recreate the aforementioned substitutions in Cbr-unc-119 and spe-44. It was found that Cbr-unc-119(zq18-like) does not have a restrictive temperature and is phenotypically comparable to the wild type allele. Similarly, zq18- and hc190-like substitutions in spe-44 do not result in substantial phenotypic variance from wild type worms. From this, we conclude that zq18- and hc190-like mutations do not universally elicit a temperature-sensitive 'toggle' in the gene that they reside. With further investigation, we hope to elucidate the relationship between highly-conserved intronic positions and temperature-sensitive gene expression.

Gillian M Stanfield et al. (2001) International C. elegans Meeting "Screening for sperm competition mutants"

Gillian M Stanfield, Anne M Villeneuve

[International C. elegans Meeting, 2001]

C. elegans hermaphrodites produce a supply of self sperm, which are normally used with extremely high efficiency (>99%). However, if a hermaphrodite mates with a male, its self sperm must compete with any transferred male sperm to fertilize oocytes. In this situation, sperm provided by the male are used preferentially(1). Male sperm precedence requires sperm motility(2) and involves displacement of self sperm from the hermaphrodite spermatheca. However, successive mating experiments have shown that sperm from different males do not exhibit any precedence relationship, indicating that sperm usage is not dictated by a "last in" mechanism of placement within the spermatheca and that there is a bona fide difference between hermaphrodite sperm and male sperm(1,3). Male sperm precedence is nearly complete: after mating, very few if any self progeny are produced until a mated hermaphrodite runs out of transferred male sperm. The strength of this effect provides a robust assay for alterations in the normal pattern of sperm usage. To identify factors that confer male sperm advantage, we are screening for mutants that do not show the normal precedence of male sperm. Specifically, we are assaying males from mutagenized him-5 lines for lack of precedence in crosses to spe-8; dpy-4 hermaphrodites. spe-8 hermaphrodites are defective in sperm activation, but spe-8 self sperm can be trans - activated upon mating to a male; dpy-4 provides a readily-scored marker for self vs. cross progeny. We expect that sperm precedence-defective males should produce sperm that are capable of fertilizing oocytes but do not suppress the production of hermaphrodite self progeny. Matings to such males should result in a mixture of self and cross progeny, and the relative numbers of self and cross progeny produced should be dependent on the numbers of each sperm type present within the hermaphrodite spermatheca. Sperm precedence mutants should be distinct from the previously described spe and fer mutants, which show defects in various aspects of spermatogenesis and/or fertilization(4). 1. Ward, S. and J.S. Carrel, Dev Biol 73 : 304-321, 1979. 2. Singson, A., K.L. Hill and S.W. L’Hernault, Genetics 152 : 201-208, 1999. 3. LaMunyon, C.W. and S. Ward, Experientia 51 :817-823, 1995. 4. L’Hernault, S.W., Spermatogenesis (p.271-294) in C. elegans II , 1997.

Doreen R Glodowski et al. (2004) East Coast Worm Meeting "Functional Characterization of the Vertebrate Homologs of LIN-10: Mint 1, 2, and 3"

Doreen R Glodowski, Bonnie L Firestein, Christopher Rongo

[East Coast Worm Meeting, 2004]

The C. elegans protein, LIN-10, functions in neurons to direct postsynaptic localization of AMPA-type glutamate receptors (AMPARs) and in epithelial cells to direct basolateral localization of the epidermal growth factor receptor (EGFR) protein, LET-23 1,2 . There are three vertebrate homologs of LIN-10: Mint 1, 2, and 3. Sequence comparison between LIN-10 and the Mint proteins, which revealed the presence of a highly divergent amino terminal region, led to the hypothesis that LIN-10 evolved in higher eukaryotes into a family of proteins with distinct cellular functions mediated by their amino terminal domains. To test this hypothesis, the activities of each Mint protein were assayed in lin-10 mutant strains of C. elegans , which display distinct phenotypes for mislocalization of AMPARs and EGFRs. Significantly, of the Mint proteins, only Mint 2 rescued mislocalization of AMPARs, demonstrating that the Mint proteins have distinct activities. Experiments testing the ability of each Mint protein to rescue mislocalization of EGFRs are ongoing and results will be presented. Further functional characterization of the Mint proteins is being performed in a vertebrate system by observing the effects of altered expression of Mint 1 and/or Mint 2 in cultured hippocampal neurons. Preliminary experiments show that a snRNA-mediated approach 3 can be used to attenuate expression of Mint 1 and/or Mint 2 in cultured hippocampal neurons. 1. Rongo, C., Whitfield, C.W., Rodal, A., Kim, S.K., and Kaplan, J.M. (1998) Cell: 94, 751-759. 2. Whitfield, C.W., Benard, C., Barnes, T., Hekimi, S., and Kim, S.K. (1999) Mol Biol Cell: 10, 2087-2100. 3. Akum, B.F., Chen, M., Gunderson, S.I., Riefler, G.M., Scerri-Hansen, M.M., and Firestein, B.L. (2004) Nature Neurosci: 2, 145-152.

Baldi, Christopher C. et al. (2011) International Worm Meeting "A Bias Caused by Ectopic Development Creates Sexually Dimorphic Sperm In Nematodes."

Ellis, Ronald E., Viviano, Jeffrey, Baldi, Christopher C.

[International Worm Meeting, 2011]

LaMunyon and Ward identified an intriguing sexual dimorphism in androdioecious nematodes - the hermaphrodites produce smaller sperm than the males. In addition, they showed that larger male sperm compete better for fertilization. These results suggested that selection had created this dimorphism in sperm size. When we used RNA interference to create C. remanei XX animals that develop as hermaphrodites (Baldi and Ellis 2009), their sperm were much smaller than those of C. remanei males. Since these animals do not produce sperm in the wild, this sexual dimorphism cannot have been created by selection. Thus, we propose that it occurs in nematodes as the result of a developmental bias. To identify the cause of this developmental bias, we are studying mutations that affect sex determination in C. elegans. First, tra-2; xol 1 XX males make large sperm like normal XO males, so sperm size is not determined by the ratio of X chromosomes to autosomes. Second, fem-3(q96) XX animals, which do not make oocytes, produce sperm that are about 25% larger than those of normal XX hermaphrodites. This result and additional studies imply that the production of oocytes by the germ line slightly decreases sperm size, perhaps because they compete for resources. Third, mutations that completely transform the somatic tissues of XX animals into male fates cause a large increase in sperm size, but mutations that cause only a partial transformation have weaker effects. Thus, we propose that several somatic tissues cooperate to nurture larger sperm in males than in hermaphrodites. These results imply that hermaphrodites produce smaller sperm than males because their bodies are not adapted for nurturing larger sperm. Hence the ectopic production of sperm in female bodies can account for this sexual dimorphism in size. We suspect that the ectopic expression of other developmental programs also alters traits during evolution. Finally, phylogenetic studies show that males from gonochoristic species make larger sperm than either males or hermaphrodites from androdioecious ones. Thus, we believe that incipient hermaphroditic species make sexually dimorphic sperm because of a developmental bias. Over time, selection further reduces sperm size in both sexes.

Herman T et al. (1995) International C. elegans Meeting "CLONING OF TWO GENES INVOLVED IN VULVAL MORPHOGENESIS AND FERTILITY"

Horvitz HR, Herman T

[International C. elegans Meeting, 1995]

We are interested in how a sheet of epithelial cells can invaginate to form a tube, such as occurs during gastrulation and organ formation in nearly all animals. The invagination of the C. elegans hermaphrodite vulva, a tube connecting the uterus to the outside, is a simple example of this process; it requires only the vulval cells themselves and a single gonadal cell, the anchor cell.1,2 We screened for mutants with a defective vulval invagination but normal vulval cell lineages. From 12,000 haploid genomes screened, we isolated 26 mutations, which we placed into eight complementation groups. Seven appear to define new genes, sqv-1 to 7 (squashed vulva), and alleles of the eighth fail to complement spe-2(mn63). The strongest alleles of all eight genes result in identical vulval phenotypes: a wild-type division pattern and detachment from the cuticle, but indistinct separation between the anterior and posterior halves of the invagination from the late L3 stage onward. We have cloned sqv-3 and spe-2, both of which map to areas sequenced by the genome project. sqv-3 is predicted to encode a homolog of mammalian beta-1,4 galactosyltransferase, an enzyme localized to the trans Golgi, where it adds galactose to proteins, many of which are then transported to the cell surface. The predicted SPE-2 protein has no similarity to known proteins but may contain a transmembrane domain. Strong alleles of each Sqv gene also cause sterility, despite the presence of sperm, oocytes and an apparently wild-type gonad. Of all the Sqv mutants, only spe-2 mutants are specifically sperm- defective (Spe): they are self-sterile but have wild-type progeny when mated to N2 males and spe-2 progeny when mated to spe-2 heterozygote males.3 One possibility is that the sterile phenotype results from defects in sperm-oocyte interactions, and that SPE-2 acts in sperm and SQV-1 to 7 act in oocytes; SQV-3 may glycosylate a SQV protein required in oocytes. We are using Sam Ward's method of artificial insemination4 to test whether sqv-1 to 7 may be oocyte-defective, which would implicate the Sqv genes in fertilization. This model might then be extended: mutations in the Sqv genes may disrupt a similar interaction among vulval cells and/or the anchor cell. We are now staining animals with antibodies against bacterially-expressed SQV-3 and are starting to make antibodies against SPE-2. 1. Kimble (1981). Dev. Biol. 87: 286. 2. Thomas et al. (1990). Cell 62: 1041. 3. Sigurdson et al. (1984). Genetics 108: 331. 4. LaMunyon and Ward (1994). Genetics 138: 689.