Chavez, Daniela et al. (2017) International Worm Meeting "Sex, Secretion, Muscle, and Males."

Stanfield, Gillian, Chavez, Daniela

[International Worm Meeting, 2017]

To successfully complete the journey to fertilize an oocyte, sperm rely on extracellular cues to direct their development and migration. In the final stages of maturation, during a process called sperm activation, sperm become polarized and develop a pseudopod used to crawl towards oocytes. Establishment of an extracellular signaling environment that spatially and temporally regulates activation is critical to fertility, as males with prematurely activated sperm are infertile. Key components of this regulation are the serine protease TRY-5 and a protease inhibitor, SWM-1. TRY-5 is a seminal fluid protein that is stored in the vas deferens and transferred to hermaphrodites, where it activates sperm. In the absence of SWM-1, TRY-5 spreads into the seminal vesicle, where sperm are stored, resulting in premature activation. We are investigating how TRY-5 is regulated by SWM-1 to ensure that sperm gain motility at the right place and time. We have discovered that somatic tissues create the sperm signaling environment that precisely controls the location and timing of activation. By analyzing a swm-1 transcriptional reporter and a SWM-1::mCherry knock-in, we determined that swm-1 is expressed by body wall muscle cells and cuboidal cells in the vas deferens while SWM-1 protein is present not only in the vas deferens but also in the seminal vesicle, surrounding sperm. SWM-1 is also present in the pseudocoelom, where it is taken up into coelomocytes. Surprisingly, tissue-specific expression suggests that muscle-derived SWM-1 is sufficient to rescue the premature activation of swm-1 mutants, but that vas deferens-derived SWM-1 fails to rescue. Our data suggest a model in which SWM-1 is secreted from muscle into the pseudocoelom and then taken up by the gonad where it attenuates TRY-5. Indeed, removing the secretion signal from SWM-1 caused it to accumulate in muscle, where it no longer rescues activation. These data show that we have identified a soma-to-germline signal critical to fertility and have determined that muscle is a key regulator of sperm cell success. Interestingly, uptake of proteins from the pseudocoelom is not specific to SWM-1, as extragonadal sources of mCherry and GFP also enter the gonad. Furthermore, vas deferens-specific mCherry is found in the pseudocoelom, thus, exchange of proteins into and out of the gonad is apparently not limited to SWM-1, and could be a general phenomenon. In ongoing work, we seek to determine whether swm-1 plays a second role within the hermaphrodite to promote reproductive success. SWM-1 is present in hermaphrodites, where it has a small but measurable effect on sperm activation. Additional SWM-1 is transferred in seminal fluid, but its role in this context is unknown. We are analyzing whether absence of SWM-1, in either the hermaphrodite or seminal fluid, affects sperm functions including migration, fertilization, or competition.

Chavez, Daniela et al. (2011) International Worm Meeting "A sperm competition mutant with defects in sperm motility."

Stanfield, Gillian, Chavez, Daniela

[International Worm Meeting, 2011]

Although males are not necessary for reproduction in C. elegans, if mating occurs then male sperm win: cross progeny are produced to the exclusion of self progeny. We would like to understand the mechanistic basis for male sperm precedence. It has been shown previously that precedence is due to an intrinsic characteristic of male sperm cells, that it requires motility, and that it correlates with the larger size of male sperm. Using a genetic screen for males with reduced precedence, we identified a mutant, me69, that has defects in sperm usage under conditions of competition with wild-type sperm. me69 male sperm show reduced precedence when they compete with wild-type hermaphrodite sperm. However, me69 hermaphrodites have normal self fertility, arguing against a generalized spermatogenesis defect. Interestingly, me69 sperm show a normal size distribution. me69 male sperm show motility defects; after transfer to the hermaphrodite, sperm accumulate poorly in the spermatheca. We are analyzing their migration in more detail to determine whether this difference arises from poor targeting, poor retention, or some other alteration in motility. We have identified a candidate gene encoding a divergent kinase that is expressed in sperm. A deletion mutant shows migration and precedence defects similar to those of me69. We are determining the localization of this kinase and investigating the possibility that either its levels or localization may be different in males vs. hermaphrodites.

Chavez, Daniela et al. (2015) International Worm Meeting "Regulation of C. elegans Sperm Motility By Protease Signaling."

Chavez, Daniela, Stanfield, Gillian

[International Worm Meeting, 2015]

C. elegans sperm undergo rapid cellular morphogenesis to develop into motile, fertilization-competent cells. During this process, termed sperm activation, cells reorganize their cytoskeleton to generate a pseudopod used to crawl towards oocytes. Sperm activation is regulated by the serine protease, TRY-5, and the serine protease inhibitor, SWM-1. TRY-5 is a seminal fluid protease transferred to hermaphrodites during mating and has the characteristics of a male sperm activation factor. In swm-1 mutant males, sperm become prematurely activated and are inefficiently transferred to hermaphrodites during mating, resulting in reduced male fertility. These previous studies suggest a model in which SWM-1 delays sperm activation by inhibiting TRY-5 protease activity from cleaving sperm cell membrane factors to initiate activation. To test this model, we have sought to determine the relationship between SWM-1 and TRY-5. It was previously shown that TRY-5 is expressed in the male somatic gonad in cells of the seminal vesicle, valve and vas deferens. We now have shown that swm-1 is co-expressed with try-5 in a subset of vas deferens cuboidal cells and that SWM-1::mCherry and TRY-5::GFP co-localize in vesicles within these cells. We also have found that swm-1 is expressed in extra-gonadal somatic cells near the seminal vesicle, and is not expressed in the valve. These data are consistent with SWM-1's inhibiting TRY-5 activity, but also suggest an alternative model where SWM-1 indirectly inhibits TRY-5, perhaps by regulating the release of TRY-5 vesicles. To further dissect the relationship of SWM-1 and TRY-5, we are analyzing the functional domains of the proteins. SWM-1 is predicted to have two trypsin inhibitor-like (TIL) domains, and previous studies have suggested that these domains may have distinct functions. To directly test the role of SWM-1 TIL domains in regulating sperm activation, we are generating precise endogenous gene edits of the predicted reactive site residues of SWM-1. Similarly, we are testing the predicted catalytic residues of TRY-5 for their role in activation. In ongoing studies, we are testing our models for SWM-1 regulation of TRY-5 activity or release by expressing swm-1 and try-5 in specific tissues and visualizing the dynamics of vesicle release from the vas deferens during mating.

Chavez, Daniela et al. (2013) International Worm Meeting "Timing is everything: dissecting the male sperm activation pathway."

Smith, Joseph, Snow, Angela, Stanfield, Gillian, Chavez, Daniela

[International Worm Meeting, 2013]

C. elegans sperm undergo rapid morphological changes to develop into polarized cells capable of migrating to and fertilizing an oocyte. During this process, termed sperm activation, the cells reorganize their cytoskeleton to generate a pseudopod, allowing them to crawl. Sperm activation is regulated by a serine protease, TRY-5, and a serine protease inhibitor, SWM-1. swm-1 mutant males contain prematurely activated spermatozoa, which are transferred inefficiently to hermaphrodites. Thus, SWM-1 is required for the temporal regulation of sperm activation and is essential to male fertility. TRY-5 is transferred in seminal fluid to a hermaphrodite and has the characteristics of a male sperm activation signal. Therefore, our model for male sperm activation is that SWM-1 directly or indirectly inhibits TRY-5 from activating spermatids within the male. Examination of the relationship between TRY-5 and SWM-1 is key to testing this model and understanding how sperm activation is regulated. To gain insight into the male sperm activation pathway, we are performing experiments to identify the location of SWM-1 expression and to analyze the functional domains of SWM-1 and TRY-5. We have previously shown TRY-5 to be expressed in the male somatic gonad in cells of the seminal vesicle, valve, and vas deferens. Similarly, we have found HA-tagged SWM-1 to be expressed in and likely secreted from a subset of vas deferens cuboidal cells, near the site of TRY-5 expression. In swm-1 mutants, TRY-5 becomes localized to the seminal vesicle. Therefore, SWM-1 and TRY-5 localization is consistent with our model in which SWM-1 inhibits TRY-5 from prematurely activating sperm. SWM-1 has two trypsin inhibitor-like (TIL) domains. We are genetically testing the necessity and sufficiency of each TIL domain for sperm activation. Preliminary results suggest that the C-terminal TIL domain plays a greater role in inhibiting sperm activation than the N-terminal TIL domain. Additionally, we are testing necessity of the catalytic domain of TRY-5 and we have preliminary evidence that it is required for activity in vivo. In the future, we will further investigate the function of SWM-1 and TRY-5 using biochemical analysis.

Hansen, Jody et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "Size, speed, signals and sex: sperm competition in Caenorhabditis elegans"

Chavez, Daniela, Stanfield, Gillian, Hansen, Jody

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Sperm competition is known to occur in many taxa, establishing it as a pervasive influence on sexual selection. Evolutionary consequences of sperm competition may include rapid molecular evolution of reproductive proteins, coevolution of the sexes, and reproductive isolation leading to speciation. While studies of sperm competition have revealed a variety of male and female traits that can influence sperm behavior and usage, the molecular mechanisms of sperm competition are poorly understood. Even less is known about the molecular nature of how females contribute to sperm selection through cryptic female choice. C. elegans is an ideal model system to study the genetic and cellular mechanisms involved in these processes since male sperm exhibit a robust competitive advantage over hermaphrodite self sperm. Male precedence relies on intrinsic differences between male and hermaphrodite sperm and requires sperm motility. Male sperm are larger than hermaphrodite sperm, and male precedence is thought to be conferred by size.Using a genetic screen, we have identified a gene, comp-1, that regulates sperm competition in C. elegans. Loss of comp-1 function results in sperm usage, migration, and storage defects, but notably does not affect sperm size. comp-1 sperm usage defects are specific to competitive contexts, as comp-1 sperm are functionally normal when wild-type sperm are absent. Furthermore, comp-1 male sperm display similar defects when competing with wild-type male sperm, indicating that the function of comp-1 is not limited to male-hermaphrodite sperm interactions. Consistent with this idea, comp-1 is conserved in closely-related male-female Caenorhabditis species.Strikingly, the comp-1 sperm defects are attenuated in hermaphrodites that lack sperm, suggesting that the comp-1 defects in sperm function are dependent on changes in the hermaphrodite environment in response to the presence of sperm. Sperm have previously been shown to communicate with the hermaphrodite reproductive tract to promote reproductive processes, such as oocyte maturation and ovulation. Currently, we are testing the idea that the hermaphrodite reproductive environment also influences sperm competition via these known signaling pathways. Our data suggests a model in which comp-1 functions in incoming male sperm to overcome inhibitory signals generated in the hermaphrodite reproductive tract in the presence of previously-established sperm. Results from our studies of comp-1 will provide insight into mechanisms of sperm competition and cryptic female choice that in turn will shed light on the broader mechanisms of evolutionary change.

Hansen, Jody et al. (2013) International Worm Meeting "Mechanisms of Sperm Competition in C. elegans."

Chavez, Daniela, Hansen, Jody, Stanfield, Gillian

[International Worm Meeting, 2013]

In C. elegans, male sperm compete with hermaphrodite self sperm, resulting in the preferential use of male sperm. This differential fertilization success, termed male precedence, is nearly absolute. Evidence indicates that male precedence relies on intrinsic differences between male and hermaphrodite sperm. One such difference is that males have larger sperm than that of the hermaphrodite. While larger sperm size is correlated with faster crawling speeds, size does not correlate perfectly with velocity, suggesting that additional factors contribute to male precedence. By identifying sperm-specific gene products required for male precedence, we seek to understand the cellular and molecular mechanisms important for cell competition. Mutations in the kinase gene F37E3.3 lead to reduced male precedence and migration defects. While wild-type male sperm migrate rapidly to the spermathecae, mutant sperm slowly accumulate in the spermathecae. However, F37E3.3 defects are dependent on the competitive context, as mutant sperm are functional by several criteria. Critically, mutant males and hermaphrodites have normal fertility. In addition, mutant sperm appear grossly normal with properly polarized sperm structures, and their in vitro velocity is indistinguishable from the wild type. The mutant sperm migration defect is ameliorated in spe-8 hermaphrodites, which lack activated sperm and contain unfertilized oocytes. Thus, the hermaphrodite reproductive environment likely plays a role in the aberrant mutant sperm migration behaviors. Currently, we are testing two models; that unfertilized oocytes might provide a better substrate for adhesion for mutant sperm, or the presence of activated hermaphrodite sperm might have an inhibitory effect on male sperm. F37E3.3 is expressed in and displays a punctate pattern that is restricted to the cell body, but it does not associate with known sperm structures. Notably, mutant spermatids are the same size as wild-type, suggesting that mechanisms in addition to size contribute to male sperm precedence. To date, F37E3.3 is the only gene identified that specifically affects C. elegans male precedence and its characterization likely will reveal a novel mechanism for sperm competition.

Shimko, Tyler et al. (2015) International Worm Meeting "Identification of regulators of sperm motility in C. elegans males."

Chavez, Daniela, Stanfield, Gillian, Shimko, Tyler

[International Worm Meeting, 2015]

The sperm of C. elegans provide an excellent model system in which the molecular interactions governing the development of motility can be studied. C. elegans sperm, unlike their swimming mammalian counterparts, move using a crawling motion, similar to that of mammalian lymphocytes. To gain motility, sperm cells must undergo a process known as activation. Previous work has shown that males and hermaphrodites rely on distinct genetic pathways to activate their sperm, though both pathways are present in sperm from each sex. In males, two factors, SWM-1 and TRY-5, have been shown to regulate sperm activation. SWM-1 activity is required to delay activation while spermatids reside in the male seminal vesicle. TRY-5 is a sperm activation signal that is transferred to the hermaphrodite during mating. Mutants of swm-1 display premature activation of spermatids inside the seminal vesicle of the male. We previously performed a genetic screen to identify mutations suppressing the swm-1 premature activation phenotype, and recovered a number of suppressors. To identify genomic regions containing suppressor mutations, we have performed bulk segregant analysis and whole genome sequencing. Common variants, variants inconsistent with the mutagen of choice, and variants in genes not enriched in sperm were filtered out of the total variant list, leaving only mutations in genes with a relatively high likelihood of affecting the activation process. For two of our suppressors, single candidate variants have been identified. We are currently testing these variants for causality by generating deletions of candidate genes using CRISPR/Cas9-targeted mutagenesis.

Rodrigues, Pedro Reis et al. (2009) International Worm Meeting "A variety of age-dependant aggregating proteins determine lifespan."

Peters, Theodore, Gibson, Brad, Lithgow, Gordon, Hughes, Robert, Alavez, Silvestre, Rodrigues, Pedro Reis, Czerwieniec, Gregg

[International Worm Meeting, 2009]

Protein aggregation has for long been hypothesised as a determinant of lifespan. Briefly, normal cellular activity may give rise to damaged proteins causing them to become insoluble, missfold and aggregate. To test this hypothesis we adapted a protocol in order to extract insoluble proteins from synchronously aging populations of C. elegans. Proteins were separated based on their aqueous and detergent solubility and the insoluble fraction was resolubilized in 70%; formic acid. Insoluble proteins were chemically labelled, identified and quantified by liquid chromatography coupled with mass spectrometry (LC- ESI-MS/MS). We identified a range of proteins with roles in various cellular processes and possibly from a range of cellular compartments. 27%; of the proteins identified as forming aggregates have previously been shown to be important in keeping low levels of polyglutamine aggregation1. This suggests that reduced soluble levels of these proteins caused by age-related aggregation may cause increased risk of polyglutamine aggregation. We then considered whether proteins that appear to form aggregates during normal aging influenced lifespan. To test this notion we, reduced their expression in adult animals (from 4 days old) by RNA interference (RNAi). 34%; of the RNAi treatments were found to significantly extend mean lifespan in C. elegans suggesting that a variety of age-dependant aggregating proteins determine lifespan. Among the insoluble proteins, DAF-21, an ortholog of the mammalian HSP-90, showed age-dependant aggregation and is being used as a marker to study the role of several molecular pathways in protein aggregation. Taken together our results suggest that protein aggregation may play a common and key role in aging and age-related disease. 1 - Nollen, E., Garcia, S., Haaften, G., Kim, S., Chavez, A., Morimoto, R., Plasterk, R., Genome-wide RNA interference screen identified previously undescribed regulators of polyglutamine aggregation. Proc Natl. Acad. Sci., 101, 6403-6408, 2004.

Thamsen, Maike et al. (2009) International Worm Meeting "Monitoring oxidative stress in aging C.elegans."

Jakob, Ursula, Thamsen, Maike, Knoefler, Daniela

[International Worm Meeting, 2009]

Aging is a complex physiological process and numerous aging theories have been proposed. One of the leading models is the free radical theory of aging, which suggests that the accumulation of reactive oxygen species (ROS), like superoxide (O2-) and hydrogen peroxide (H2O2), causes protein, lipid and DNA damage and leads to the observed age-related decline of cells and tissues. To directly monitor the onset and extent of oxidative stress during the lifespan of C. elegans, we utilize two complementary approaches. In the first approach, we use the fluorescent H2O2-sensor protein HyPer (Belousov, 2006) to evaluate the accumulation of endogenous H2O2 in the muscle cells of C. elegans. This ratiometric sensor protein has two excitation maxima, which substantially change in the presence of H2O2. With this tool, we are now able to determine and monitor endogenous H2O2 levels over the whole life span of a worm population. In the second approach, we use a highly quantitative mass spectrometry based thiol trapping technique termed OxICAT to identify the protein targets of oxidative stress in aging animals. This technique allows us not only to detect and quantify oxidative thiol modifications in hundreds of different proteins in a single experiment but enables us to identify the affected proteins and to define their redox-sensitive cysteine(s). Using this technique we were able to determine the redox status of numerous C. elegans proteins and monitor their age-related changes. The combination of these techniques provides us now with valuable insights into the underlying mechanism of aging and into the role that oxidative stress plays in this process. Reference: Belousov, V.V., et al., Nat Methods, 2006. 3(4). Maike Thamsen and Daniela Knoefler contributed equally to the work for this abstract.

Ellen Nollen et al. (2006) European Worm Meeting "C. elegans Models of Protein Misfolding Diseases"

Ellen Nollen, Tjakko van Ham, Ronald H. A. Plasterk.

[European Worm Meeting, 2006]

Ellen Nollen, Tjakko van Ham & Ronald H. A. Plasterk. Aggregation of misfolded proteins occurs in various age-related neurodegenerative disorders, including Parkinsons, Alzheimers, and Huntingtons disease. To understand how cells protect themselves against misfolded proteins, we search for genes that enhance or prevent protein aggregation. C. elegans strains expressing polyglutamine stretches fused to YFP with visible, age-dependend protein aggregation are used as a genetic model. Using a genome-wide RNAi screen, we have previously identified 186 genes that, when knocked down, cause premature protein aggregation. These genes include genes involved in protein synthesis, folding, degradation and RNA synthesis and processing. 1. Conversely, we performed a forward mutagenesis screen to identify genes that, when mutated, suppress age-dependent polyglutamine aggregation. For one suppressor mutant, in which aggregation is suppressed by more than 75%, we have now identified the responsible mutation. This mutation is a missense mutation in a gene encoding a protein of unknown function that is highly conserved between C. elegans and humans. Knock-down by RNAi of the same gene in wild-type worms yielded a similar reduction in aggregation, suggesting a loss-of-function mutation. We are currently further characterizing this mutant and the remaining suppressor mutants. In addition, to establish whether the genes we have identified are specific for polyglutamine aggregation or whether they comprise of a general protein homeostatic buffer, we have developed a worm model for aggregation of alpha synuclein, which occurs in Parkinson''s disease. Altogether our results will provide insight into cellular protection against misfolded proteins and yield targets for therapy against protein misfolding diseases.. 1Nollen E.A.A., Garcia S.M., van Haaften G., Kim S., Chavez A., Morimoto R.I., Plasterk R.H. (2004) Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc. Natl. Acad. Sci. U.S.A. 101(17):6403-8.