Untangling Reproductive Success: Caenorhabditis Nematodes as a Model System for Fertilization Katja R. Kasimatis, Megan J. Moerdyk-Schauwecker, and Patrick C. Phillips Institute of Ecology and Evolution, University of Oregon

Reproductive Health is a World-Wide Goal What Comprises the Male Reproductive Proteome? What Determines Fertilization Success? Goal: To characterize the complete set of sperm proteins (sperm proteome) in Goal: To quantify the relative importance of post-insemination dynamics in determing World-Wide Female Infertility1 US Infertility Rate2

World 12 C. elegans and C. remanei and analyze the molecular evolution. total reproductive success using experimental evolution.

Latin American and Caribbean

East Asia and Pacific 10 tility Asia, South e r Sperm Collection: Shredder v5.0 male reproductive success = successfully mate + successfully fertilize North Africa and Middle East 8 ercent In f Sub−Saharan Africa P female choice cryptic choice Central/Eastern Europe and Central Asia 1 mm 6 Female & & North America and Western Europe Male

0 10,000,000 20,000,000 1982 1988 1995 2002 2010 male-male competition sperm competition Estimated Number of Women Year

Sexual Selection • Reproductive health includes the “capability to reproduce ... and the freedom to display preference decide if, when, and how often” (WHO) competition • In US, 90% of infertility examinations are initiated in female partners alone, while males mating❤ account for ~33% of all infertilities2,3 • Impaired fecundity and infertility are characterized by abnormal sperm and poor 500 um 10 um 500 um sperm semen quality competition • These same sperm traits are related to male-specific health issues, such as testis and prostate cancers3 cryptic • CDC now considers infertility a disease choice

Understanding the molecular basis of infertility is important for • Microfluidic technique that allows for worms to be manipulated on a micron scale fertilization❤ diagnosing and treating not only infertility, but also male-specific health issues. • Dissects 15-20 males per device • Collect 100s of sperm cells per male Natural Selection 1Mascarenhas et al. (2012) PLoS Medicine. 2National Center for Health Statistics (2013). 3CDC (2013). Sperm Activation Experimental evolution is a powerful way to study the action of selection and its phenotypic and genotypic consequences. • Mature sperm requires activation Caenorhabditis Nematodes as a Model System activation • Pseudopods form and membranous organelles Spermatid Spermatozoon (MO) fuse with cell membrane Genetic Transformations for Inducible Sterility and Inducible Lethality • Function of MOs is uncharacterized Caenorhabditis nematodes are a model organism for aging, Symbol Molecular Mechanism Phenotype control • Using in vitro activation separate MO proteins developmental biology, evolutionary genetics, molecular membranous organelle degron spe-44 normal spermatogenesis proteins sperm can be separated from activated sperm proteins No Auxin ♂E normal mating proteins genetics, and neurobiology. TIR1 sperm transfered activation cell lysis

auxin degron spe-44 arrested spermatogenesis Auxin ♂E TIR1 nigoni Ub normal mating Ub Experimental Ub no sperm transfered briggsae Sperm Proteome Composition ⚥ • Free-living life-history with a preference for rotting fruit sinica 0.10 Membranous Activated Sperm ♂ normal cellular function and vegetation Organelle 20C C remanei latens • Natural variation in the strength of sexual selection 0.08 17 29 14 • Activated sperm and MO have unique sp. 33 • Three independent lineage transitions to 35C ♂ ectopic expression of PEEL-1 proteomic signatures C muscle & epidermal breakdown self-fertilizing hermaphroditism 0.06 Competitor wallacei • Major Sperm Protein most abundant death within 3 hours • One lineage transition to sperm gigantism undance tropicalis ⚥ • Identified uncharacterized, nematode- doughertyi • High quality genome assemblies available for most of 0.04 Mean A b specific proteins in MO sp. 34 the Elegans supergroup • Conservation of composition between elegans ⚥ 0.02 Experimental Design kamaaina species

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Generation{ {

act−2 act−3 act−4 act−5 pdi−1 pl−13 pl−21 pl−28 dld−1 Generation par−5

hsp−6 ps−18 ps−30 asp−5 hsp−4 t+1 NSPD gdh−1 SAMS r r r t enol−1 mdh−1 r r aagr−3 ssp−35 dnpp−1

ZK632.9 mating competition T28D9.1 F13H6.3 F37C4.5

M176.10 sperm longevitysperm offense R06C1.4 sperm defense W01D2.1 M02D8.10 HS ♀ × ♂ ♀ × ♂ + ♂ ♀E ♂E Unique to Membranous Organelle Unique to Activated Sperm Post-insemination E E E E C – + + – auxin eggs ♀C ♂C ♀ ♂ Competition 24 hours 48 hours E E

♀ × ♂ ♀ × ♂ HS E E E E ♀E ♂E – + – – Major Sperm Protein Evolution No Competition auxin eggs ♀E ♂E Hyper-Conservation of the Gene Sequence Rapid Gene Family Evolution 24 hours 48 hours CE sp34 HS mitotic transition meiotic zone seminal vas deferens cloaca 100% V 100% ♀ × ♂ ♀ × ♂ + ♂ ♀E ♂E III E E E E C msp−3 msp−40 msp−33 msp−32 msp−31 msp−152 msp−74 msp−45 msp−49 msp−50 msp−142 msp−63 msp−64 IV–β II Full Competition ♀ ♂ + – + + zone zone vesicle 95% auxin eggs C C 95% | | |||| | | || || || | | ||| || | | | ♀E ♂E

90% ig−6 24 hours 48 hours r gst−9 pld−1 gst−7 ttm−2 ptp−2 lact−5 mpk−2 mog−5 nep−12 ampd−1 IV–α btbd−10 90% II-d 0 Mb 1,154,467 Mb 85% • Mate choice is determined by pheromone signaling A CE • Mating is initiated by the insertion of the male spicule into the female, triggering the 85% 80% |||||||| |||| | || | • Capitalizing on the genetic power of C. elegans to completely isolate post-insemination II-a II-c B C D II-b • Select for sperm defensive capability and longevity ejaculation reaction sp34 | || | || || ||||| | || I ||||||| || | | | • Immature spermatids are activated to form mature, motile spermatozoa II-a IV–α IV–β I II-b II-c II-d II III V • Ancestral population is an outcrossed wild-isolate E F CBG 100% CBG • Sperm crawl up the female reproductive tract to the spermathecae and wait to fertilize V | ||| | | | || || • Evolve populations of thousands of worms for tens of generations and measure the | | | || |||| released oocytes III 95% phenotypic and genomic changes due to sexual selection CRE 90% || | | | | | • The proteomic components of sperm and seminal fluid are largely unknown as well as II-e | | ||| |||| −2 0 2 4 6 8 10 the genetic basis for fertilization 85% Relative Chromosome II Position (Mb)

Pairwise nucleotide II-f Colored up-ticks represent Chromosome II MSPs and gray We would like to thank the MacCoss Lab (UW) for mass spectrometry analysis, Nadine sequence identity. down-ticks represent Chromosome II anchors. Timmermeyer for proto-typing the shredder, and Stephen Banse for constructive advice. Nematodes are an excellent model system for untangling the components of II-f II-e III V reproductive success and analyzing how they evolve both experimentally K. Kasimatis is supported by the NIH and ARCS Oregon Chapter. and in natural populations. Interplay of functional constraint on the gene sequence and evolution at the level of the gene family. Contact: [email protected]