Proteomic Insights into Seminal Plasma and Spermatozoa Proteins of Small-Spotted Catsharks, Scyliorhinus canicula: Implications for Reproductive Conservation in Aquariums

Abstract

In the ex situ conservation of chondrichthyan species, successful reproduction in aquaria is essential. However, these species often exhibit reduced reproductive success under human care. A key aspect is that conventional sperm analyses do not provide insights into the functional competence of sperm. However, proteomics analysis enables a better understanding of male physiology, gaining relevance as a powerful tool for discovering protein biomarkers related to fertility. The present work aims to build the first proteome database for shark semen and to investigate the proteomic profiles of seminal plasma and spermatozoa from small-spotted catsharks (Scyliorhinus canicula) related to the underlying adaptations to both natural and aquarium environments, thereby identifying the reproductive impact in aquarium specimens. A total of 305 seminal plasma and 535 spermatozoa proteins were identified. Among these, 89 proteins (29.2% of the seminal plasma set) were common to both spermatozoa and seminal plasma. In the seminal plasma, only adenosylhomocysteinase protein showed differential abundance (DAP) between wild and aquarium animals. With respect to the spermatozoa proteins, a total of 107 DAPs were found between groups. Gene Ontology enrichment analysis highlighted the primary functional roles of these DAPs involved in oxidoreductase activity. Additionally, KEGG analysis indicated that these DAPs were primarily associated with metabolic pathways and carbon metabolism. In conclusion, we have successfully generated an initial proteome database for S. canicula seminal plasma and spermatozoa. Furthermore, we have identified protein variations, predominantly within spermatozoa, between aquarium and wild populations of S. canicula. These findings provide a foundation for future biomarker discovery in shark reproduction studies. However, additional research is required to determine whether these protein variations correlate with reproductive declines in captive sharks.

Keywords: Elasmobranchii; Scyliorhinidae; conservation biology; sperm; spotted dogfish.

Figure 1

Representative images of small-spotted catshark (Scyliorhinus canicula) sperm cell quality. Scale bar, 20 μm. (A) Phase-contrast image of sperm cells at 400× magnification. (B) Phase-contrast and epifluorescence merged micrographs of sperm cells stained with SYBR-14 (green) and propidium iodide (red) at 200× magnification. Green fluorescence shows live sperm, and red fluorescence indicates dead sperm.

Figure 2

Principal component analysis (PCA) (left panels) showing the separation among animal origin for the seminal plasma (A) and spermatozoa (B) of total proteins. Blue color indicates wild animals. Red color indicates aquarium animals. Dots with similar color indicate technical replicates for each source (n = 4). Heatmap with dendrograms (right panels) representing the differentially abundant proteins among seminal plasma (C) and spermatozoa (D) from aquarium (A) and wild (W) animals. The data were obtained from 4 replicates for each source. The hierarchical clustering tree is shown at the top of the heat map. The relative expression level of the differentially abundant proteins is shown on a color scale from orange representing the highest level to green representing the lowest level.

Figure 3

Differentially abundant proteins in seminal plasma and spermatozoa protein extracts from wild and aquarium samples of small-spotted catshark (Scyliorhinus canicula). (A) Venn diagram showing the number of unique proteins in seminal plasma and spermatozoa samples. (B) Number of proteins exhibiting significant differences within seminal plasma and spermatozoa between aquarium and wild groups. Visualizations of DAPs in volcano plots using Metaboanalyst are as follows: the x-axis represents logFC; the y-axis represents the −log10 of a p-value of <0.05. Proteins with logFC ≥ 1 are represented by red dots, and those with logFC ≤ −1 by blue dots. Significant DAPs are labeled with gene names. Black dots indicate proteins that did not show significant changes. (C) Volcano plot for seminal plasma proteins. (D) Volcano plot for spermatozoa proteins.

Figure 4

Protein-protein interaction networks of spermatozoa proteins differentially abundant between wild and aquarium semen annotated in the UNIPROT in cloudy catshark (Scyliorhinus torazame). One cluster of interacting proteins was identified using the STRING software (version 12. https://string-db.org, accessed on 1 March 2024) with a high confidence score. The line size indicates a high interaction score (tight lines indicate a high score > 0.7; thin lines indicate a medium score > 0.4). Each node represents a protein, whereas edges indicate the strength of the relationship between proteins (i.e., more edges indicate higher confidence). The proteins identified in small-spotted catshark spermatozoa of aquarium and wild males are shown in Table 2.