Singson, Andrew (2012) Worm Breeder's Gazette "Notable recent papers in reproductive biology"

Singson, Andrew

[Worm Breeder's Gazette, 2012]

Andrew Singson et al. (2007) International Worm Meeting "Genes required for gamete activation and fertilization."

Jean Parry, Rika Maruyama, Andrew Singson, Julie Hang

[International Worm Meeting, 2007]

Reproductive success requires that two haploid cells - sperm and egg - unite to form a diploid zygote. Despite being well described, the molecular underpinnings of fertilization are still poorly understood. We are addressing the molecular events required for reproductive success. The most significant advantage of C. elegans is the ability to isolate and maintain mutants that affect sperm or eggs and no other cells. We have focused on four classes of sterile mutants. The first class of mutants defines genes that are required for sperm activation (spermiogenesis mutants). The second class of mutants defines genes that are required by sperm to fertilize the egg (sperm function mutants). The third class of mutants defines genes required in the egg to be fertilized by sperm (egg function mutants). The last class of mutants is required in the egg to trigger development after fertilization (egg activation mutants). This work is providing us with a better understanding of the molecular mechanisms of fertilization in C. elegans. It also highlights many fundamental questions in cell biology and provides insights into the diversity of reproductive strategies.

Andrew Singson et al. (2002) West Coast Worm Meeting "Mutants that affect gamete function at fertilization"

Emily Putiri, Indrani Chatterjee, Pavan Kadandale, Brian Geldziler, Marty Nemeroff, Andrew Singson

[West Coast Worm Meeting, 2002]

The main goal of our lab is to understand the molecular mechanisms of fertilization. Initially, we have focused on the characterization of a group spe and fer genes required by sperm for fertilization. Although sterile, worms with these mutations produce morphologically wild-type sperm with normal motility that can come in contact with oocytes. Therefore mutations in these genes disrupt gamete recognition, adhesion, signaling and/or fusion. We have expanded our studies to include gene products required by the oocyte for fertilization. We are using RNAi to test candidate egg cell surface components for possible functions in fertilization. Additionally, we are conducting a genetic screen to identify conditional "egg sterile" mutants. We will report our progress on the genetic, phenotypic and molecular analysis of these genes. This work will lead to a better understanding of the specialized cell-cell interactions that occur at fertilization.

Fire A et al. (1994) Worm Breeder's Gazette "Vectorology I: New lacZ Vectors (Building a Better Gene Trap)."

Fire A, Xu SQ

[Worm Breeder's Gazette, 1994]

Vectorology I: New lacZ vectors ("building a better gene trap") Andrew Fire and SiQun Xu Carnegie Institution of Washington, Baltimore, Md 21210

Mehra A et al. (1994) Worm Breeder's Gazette "Direct Interaction Between FEM-3 and a Carboxy-Terminal Fragment of TRA-2A"

Spence AM, Heck L, Kuwabara PE, Mehra A

[Worm Breeder's Gazette, 1994]

Direct Interaction between FEM-3 and a Carboxy-Terminal Fragment Or TRA-2A Arun Mehra, Linda Heck, Patricia Kuwabara*, and Andrew Spence Dept. of Medical Genetics, University of Toronto, 1 King's College Circle, Toronto, Canada, and *MRC Laboratory of Molecular Biology, Hills Rd., Cambridge, UK

Singaravelu, Gunasekaran et al. (2009) International Worm Meeting "Isolation of Caenorhabditis elegans mutants defective in sperm function."

Shakes, Diane, Singaravelu, Gunasekaran, Singson, Andrew

[International Worm Meeting, 2009]

The functional components required during fertilization can be conveniently studied in Caenorhabditis elegans. Here we describe a screening strategy for isolation of C. elegans mutants defective in sperm function (Spe) or egg function (Egg). We mutagenized the hermaphrodite population of the genotype sem-2(n1343) Is[Pelt-7::gfp + rol-6 (su 1006)] I using Ethyl methyl sulfonate (EMS) and screened for the self sterile mutants that could be rescued by allowing them to mate with wild type males. The sem-2(n1343) genetic background prevents the worm from laying eggs and the progeny are hatched inside the body. The transgene, Pelt-7::gfp, drives the expression of gfp under elt-7 promoter, which is active in intestine. Thus, amid the mutagenized population, sterile mutants can be readily identified by the absence of fluorescing eggs and/or larvae in their uterus. In our most recent screen, we isolated twenty-four non-conditional sterile Spe mutants and eleven temperature sensitive Spe mutants. Our preliminary characterization suggests that various aspects of C. elegans reproductive biology such as sperm development, differentiation and/or functions are compromised in these mutants. A slightly modified strategy can be used to identify mutants that cause defective oogenesis or oocyte function. We are currently mapping and further characterizing these mutants with the aim of unraveling the genetic determinants behind the normal, reproductively competent sperm.

Rahimi, Sina et al. (2011) International Worm Meeting "Mapping and Characterization of Caenorhabditis elegans Mutant Defective in Sperm Function."

Singson, Andrew, Singarevelu, Gunasekaran, Rahimi, Sina

[International Worm Meeting, 2011]

The molecular identities of the proteins participating in sperm and egg interactions during fertilization are unraveled by the isolation and mapping of mutants defective in the process. Here we report our preliminary results on mapping and characterization of as38, a non-conditional sperm function defective mutant that we isolated from a recent screen induced by Ethyl Methyl Sulfonate (EMS). Sperm function is defined as the ability of a normal, developed sperm to bind and enter the oocyte to execute fertilization. We have placed as38 in "spe-9 class" of mutants which are a group of mutants known for defective sperm function. Gonad morphology and the other processes of gamete development and differentiation occur normally in as38. Although leaky, hermaphrodites of as38 show significantly lower brood size in comparison to wild type. As38 shows temperature dependent decline in brood size. Examination of the oocytes that exit spermatheca indicates the lack of sperm entry and hence the unfertilized oocytes become endomitotic. The sperm from as38 male can successfully migrate into the spermatheca of recipient hermaphrodite. However, the fertility is significantly compromised in as38 males. Sperms from dissected males show normal morphology and undergo normal in vitro activation in response to Pronase E treatment. Using two factor mapping, we have placed as38 on the right arm of the Linkage Group IV near unc-26. We are currently performing three factor mapping to clone the as38 allele.

Krauchunas, Amber et al. (2015) International Worm Meeting "spe-43 is a member of the spe-8 class of genes and is required for spermiogenesis in C. elegans."

Mendez, Ernesto, Parry, Jean, Krauchunas, Amber, Singson, Andrew

[International Worm Meeting, 2015]

Successful fertilization requires that sperm are activated prior to contacting an oocyte. In C. elegans, this activation process, called spermiogenesis, transforms round immobile spermatids into motile, fertilization-competent spermatozoa. Spermiogenesis in males is dependent on the TRY-5 protease pathway, whereas in hermaphrodites it is controlled by the SPE-8 pathway. Here we describe the phenotypic and genetic characterization of spe-43, a new component of the SPE-8 pathway; spe-43 hermaphrodites are self-sterile, while spe-43 males show wild-type fertility. Consistent with other members of the spe-8 class, spe-43 hermaphrodite sperm can be trans-activated by male seminal fluid. However, when exposed to Pronase to activate the sperm in vitro, spe-43 spermatids form long rigid spikes radiating outward from the cell periphery instead of forming a motile pseudopod. We also find that spe-43 male sperm are smaller and move more slowly than sperm from N2 males. Yet these sperm are not only capable of successful fertilization, they are also able to outcompete hermaphrodite sperm from fertile animals, suggesting that size and speed are not the only factors that provide male sperm with their competitive advantage over hermaphrodite sperm. Using a combination of recombinant and deletion mapping and whole genome sequencing we identified a C>T mutation in F09E8.1 as the molecular lesion in spe-43(eb63). Transgenic rescue experiments confirm that F09E8.1 is the spe-43 gene. SPE-43 is predicted to exist in two isoforms; one isoform appears to be a transmembrane protein with a large extracellular domain while the other is predicted to be a secreted protein.

Wendy Wong et al. (2006) European Worm Meeting "The Integrative Interaction Map for Caenorhabditis elegans"

Wendy Wong, Andrew Fraser

[European Worm Meeting, 2006]

. Wendy Wong and Andrew Fraser. C. elegans is a popular model system for the systematic analysis. However, relatively little is known for its networks of genetic interactions. We hereby construct an integrative interaction map for C. elegans by integrating diverse functional genomics Datasets. We will also present some of the novel tools in querying the interaction map for answering powerful biological hypotheses.

Marcello, Matthew R et al. (2011) International Worm Meeting "Development of a C. elegans Spermiogenesis and Sperm Function Protein Interaction Network."

Marcello, Matthew R, Singson, Andrew, Singaravelu, Gunasekaran, Druzhinina, Marina

[International Worm Meeting, 2011]

The proper temporal and spatial organization of proteins during the spermatid to spermatozoa transition is necessary for the spermatozoa to function properly during fertilization. However, the protein-protein interactions necessary for ensuring spermiogenesis and sperm function during fertilization are not well understood. Sperm-enriched genes necessary for these processes are underrepresented in high throughput protein-protein interaction studies because of the low abundance of these proteins relative to the entire worm proteome. The purpose of this study was to generate a protein-protein interaction network for the proteins that are necessary for spermiogenesis and sperm function in C. elegans. The development of this protein interaction network will allow for the identification of novel interactions between proteins necessary for these processes and will also further our understanding of how spermiogenesis and sperm function are coordinated. To identify interacting proteins, we performed pair-wise split-ubiquitin yeast two-hybrid analysis of the full-length gene products of genes that have a known function in spermiogenesis (fer-1, spe-4, spe-6, spe-8, spe-12, spe-19, spe-27, and spe-29) or sperm function during fertilization (fer-14, spe-9, spe-13, spe-38, spe-41, and spe-42). Our results indicate that for some genes that that interact genetically there are physical interactions between their gene products. For example, spe-6 is known to interact with spe-19 and spe-29 genetically; our results indicate that SPE-6 also physically interacts with SPE-19 and SPE-29. We also identified novel protein-protein interactions, including interactions between SPE-8 and SPE-19 and SPE-9 and SPE-19. We represented the network topology using Power Graphs analysis rather than standard representations so as to remove redundancy and simplify network visualization. A future goal is to develop an integrated network model that incorporates additional datasets, including genetic interactions, expression profiles, localization data, and additional protein interaction data, along with the protein-protein interaction network we have generated. The integrated network of spermiogenesis and sperm function proteins will provide a robust source for generating hypotheses about how to assemble functional spermatozoa capable of fertilization.