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, 19 (1), 593

Proteomic and Evolutionary Analyses of Sperm Activation Identify Uncharacterized Genes in Caenorhabditis Nematodes

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Proteomic and Evolutionary Analyses of Sperm Activation Identify Uncharacterized Genes in Caenorhabditis Nematodes

Katja R Kasimatis et al. BMC Genomics.

Abstract

Background: Nematode sperm have unique and highly diverged morphology and molecular biology. In particular, nematode sperm contain subcellular vesicles known as membranous organelles that are necessary for male fertility, yet play a still unknown role in overall sperm function. Here we take a novel proteomic approach to characterize the functional protein complement of membranous organelles in two Caenorhabditis species: C. elegans and C. remanei.

Results: We identify distinct protein compositions between membranous organelles and the activated sperm body. Two particularly interesting and undescribed gene families-the Nematode-Specific Peptide family, group D and the here designated Nematode-Specific Peptide family, group F-localize to the membranous organelle. Both multigene families are nematode-specific and exhibit patterns of conserved evolution specific to the Caenorhabditis clade. These data suggest gene family dynamics may be a more prevalent mode of evolution than sequence divergence within sperm. Using a CRISPR-based knock-out of the NSPF gene family, we find no evidence of a male fertility effect of these genes, despite their high protein abundance within the membranous organelles.

Conclusions: Our study identifies key components of this unique subcellular sperm component and establishes a path toward revealing their underlying role in reproduction.

Keywords: Molecular evolution; Nematodes; Proteomics; Spermiogenesis.

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The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Spermiogenesis in nematodes. a In un-activated spermatids, membranous organelles (shown in teal) migrate to the cell periphery, while Major Sperm Protein (shown in gray) is distributed throughout the cell. Upon sperm activation, Major Sperm Protein forms the pseudopod of the cell and is used to crawl, while the membranous organelles fuse with the cell membrane and release their contents into the extracellular space. b Diagram of the sperm collection processes. Un-activated spermatid proteins were collected by concentrating spermatids collected using microfluidic dissection (see Fig. 2) and lysed to release proteins. For the activated proteome, un-activated spermatids were first collected using a male crushing technique and then concentrated. The supernatant before sperm activation represents a control for cell lysis. Spermatids were activated in vitro by changing the intracellular pH. The supernatant after activation represents the proteins released during membranous organelle fusion. The activated sperm cells were lysed and the membranes pelleted. The supernatant after cell lysis represents the proteins associated with the activated sperm body
Fig. 2
Fig. 2
Schematic of The Shredder. a The Shredder is a microfluidic dissection device with a single worm loading arena, a needle insertion, a sperm filtration and collection arena, and two flush channels. b The male loading arena. The bifurcating design sequentially loads males into the dissection channel. c The male dissection channel. Males are pushed into the channel from the loading arena and sperm cells are flushed out the right. The needle channel is separated from the male dissection channel by a thin filament of PDMS, which creates a water-tight seal around the needle. d The sperm filter (10 um) prevents collection of non-sperm components. e Males in the loading arena for sequential loading into the dissection channel. f Dissected male and released spermatids (indicated by the triangle) for collection
Fig. 3
Fig. 3
Proteomic characterization of the membranous organelle and activated sperm proteomes in C. elegans. a The two proteomes were distinct, with 17 proteins found only in membranous organelles and 14 proteins found only in activated sperm. b The 15 most abundant proteins identified in the membranous organelles. Proteins unique to membranous organelles (highlighted in teal) include the Nematode-Specific Peptide family, group D (NSPD) as well as several housekeeping gene families. c The 15 most abundant proteins identified in activated sperm. The proteins unique to activated sperm (highlighted in teal) are predominantly involved in energy production. Protein abundance is shown as the relative mean normalized spectrum abundance frequency
Fig. 4
Fig. 4
The evolution of the Nematode-Specific Peptide family, group D (NSPD) across the Caenorhabditis elegans Supergroup. Listed for each species are: the number of gene copies annotated, the genomic location (Roman numerals represent chromosome level assemblies and numbers represent scaffolds), the mode coding sequence length in base pairs (n = number of gene copies of said length), the mean amino acid sequence identity between paralogs, and the alignment-wide estimate of the ratio of non-synonymous to synonymous substitutions (ω). The complete gene annotation list is provided in Additional file 3 and the sequence alignments are given in Additional file 4
Fig. 5
Fig. 5
The evolution of the Nematode-Specific Peptide family, group F (NSPF) across the Elegans Supergroup. The orthologous nspf-1 and nspf-3 genes are shown in orange on the chromosome or scaffold to which they locate. The Chromosome IV gene anchors used to determine synteny are shown. For each orthologous group the mode coding sequence length (in base pairs), the mean amino acid sequence identity, and the alignment-wide estimate of the ratio of non-synonymous to synonymous substitutions (ω) are shown. The C. elegans orthologs are excluded from the mean identity and ω estimates as they show distinctly different patterns of evolution. The complete gene annotation list is provided in Additional file 3 and the sequences alignments are given in Additional file 6
Fig. 6
Fig. 6
Functional assays of the NSPF gene family in C. elegans male fertility. a In a non-competitive sperm setting, knockout males (orange) do not produce significantly fewer progeny than control males (gray) when given an excess of females with which to mate (t = − 0.81, df = 26.0, p = 0.42). b In a competitive sperm setting, knockout males (orange) do not produce significantly fewer progeny than control wildtype males (z = − 0.12, p = 0.90) nor do they produce a significant deviation from 50% of the total progeny production (proportions test: χ2 = 1.27, df = 1, p = 0.26, C.I. of progeny produced = 27.4–55.9%). All fecundity data are provided in Additional file 7

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