Recurrent Co-Option and Recombination of Cytokine and Three Finger Proteins in Multiple Reproductive Tissues Throughout Salamander Evolution.

Damien B Wilburn,Pamela W Feldhoff,Brian C Searle,Richard C Feldhoff,Christy L Kunkel

doi:10.3389/fcell.2022.828947

Abstract

Reproductive proteins evolve at unparalleled rates, resulting in tremendous diversity of both molecular composition and biochemical function between gametes of different taxonomic clades. To date, the proteomic composition of amphibian gametes is largely a molecular mystery, particularly for Urodeles (salamanders and newts) for which few genomic-scale resources exist. In this study, we provide the first detailed molecular characterization of gametes from two salamander species (Plethodon shermani and Desmognathus ocoee) that are models of reproductive behavior. Long-read PacBio transcriptome sequencing of testis and ovary of both species revealed sex-specific expression of many genes common to vertebrate gametes, including a similar expression profile to the egg coat genes of Xenopus oocytes. In contrast to broad conservation of oocyte genes, major testis transcripts included paralogs of salamander-specific courtship pheromones (PRF, PMF, and SPF) that were confirmed as major sperm proteins by mass spectrometry proteomics. Sperm-specific paralogs of PMF and SPF are likely the most abundant secreted proteins in P. shermani and D. ocoee, respectively. In contrast, sperm PRF lacks a signal peptide and may be expressed in cytoplasm. PRF pheromone genes evolved independently multiple times by repeated gene duplication of sperm PRF genes with signal peptides recovered through recombination with PMF genes. Phylogenetic analysis of courtship pheromones and their sperm paralogs support that each protein family evolved for these two reproductive contexts at distinct evolutionary time points between 17 and 360 million years ago. Our combined phylogenetic, transcriptomic and proteomic analyses of plethodontid reproductive tissues support that the recurrent co-option and recombination of TFPs and cytokine-like proteins have been a novel driving force throughout salamander evolution and reproduction.

Highlights

Sexual reproduction is a hallmark of most animal life cycles and is the process by which progeny are generated from the recombination of two genomes, usually from individuals of the same species
We provide the first detailed molecular description of salamander gametes based on long-read transcriptomic analyses of plethodontid ovary and testis, paired with proteomic analysis of plethodontid sperm from the model species P. shermani and D. ocoee
Using the gonad transcriptome supplemented with mental gland cDNA sequences as a search database, we identified a total of 7,287 peptides for 1,648 protein groups in P. shermani sperm and 5,308 peptides for 1,157 protein groups in D. ocoee sperm

Summary

Introduction

Sexual reproduction is a hallmark of most animal life cycles and is the process by which progeny are generated from the recombination of two genomes, usually from individuals of the same species. Published structural and biochemical studies have begun to provide insight into the molecular basis of egg-sperm recognition in marine invertebrates, fishes, birds, and mammals (Ichikawa et al, 2016; Raj et al, 2017; Herberg et al, 2018; Wilburn et al, 2018), but among vertebrates substantially less attention has been given to reptiles and amphibians This is especially true for salamanders, where to date there have been no studies characterizing the molecular composition of salamander gametes. We present the first molecular characterization of salamander gametes for two species that are established models of reproduction and prezygotic mating barriers

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Results

Conclusion