# First insight into the regulatory role of the glycoprotein hormones GPA2 and GPB5 in the small-spotted catshark (Scyliorhinus canicula L.) during spermiogenesis.

Fabian Jeanne, Stanislas Pilet, Benoît Bernay, Pascal Sourdaine

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Fabian Jeanne, Stanislas Pilet, Benoît Bernay, Pascal Sourdaine. First insight into the regulatory role of the glycoprotein hormones GPA2 and GPB5 in the small-spotted catshark (Scyliorhinus canicula L.) during spermiogenesis. Journal of Proteomics, 2026, 327, pp.105627. 10.1016/j.jprot.2026.105627. hal-05534349

HAL Id: hal-05534349

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# First insight into the regulatory role of the glycoprotein hormones GPA2 and GPB5 in the small-spotted catshark (*Scyliorhinus canicula* L.) during spermiogenesis.

Fabian Jeanne<sup>a</sup>, Stanislas Pilet<sup>a</sup>, Benoît Bernay<sup>b</sup>, Pascal Sourdaine<sup>a,∗</sup>

## ARTICLE INFO

**Keywords:**

GPA2

GPB5

Thyrostimulin

Spermiogenesis

Chondrichthyes

Elasmobranchs

## ABSTRACT

Glycoprotein hormones alpha 2 (GPA2) and beta 5 (GPB5) are considered to be ancestral members of the glycoprotein hormone (GPH) family. Despite their potential roles in regulating thyroxine production, the immune response, and ovarian function, their roles in the testis remains largely unknown. To further explore this regulation, catshark testicular explants containing late spermatid stages were treated *in vitro* with recombinant GPA2, GPB5 and single-chain GPB5#GPA2. The comparative proteomic analysis of the treated explants revealed that ScGPB5 and ScGPB5#ScGPA2 were the most effective, modulating abundance of 449 and 525 proteins, respectively. In contrast, ScGPA2 only modulated 212 proteins. Among the differentially abundant proteins (DAPs), 103 showed substantial abundance changes in response to at least one treatment, with mean Log2ratios of 2.5 and -2.5. Manual functional annotation linked these proteins to spermiogenesis and immunity. Key proteins and candidates involved in spermiogenesis were highlighted, including SPINK2, which is involved in acrosome protection, and LRRC69, which is related to human spermatogenic failure. These results reinforce the hypothesis of a GPA2/GPB5 paracrine regulation of catshark spermiogenesis. They also underscore the necessity of functional and comparative studies to elucidate the physiological functions of GPA2/GPB5 across a broad spectrum of taxa, from non-vertebrates to mammals.

**Significance:** To our knowledge, this study is the first to report the effects of stimulation with recombinant ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2 on vertebrate testicular tissue *in vitro*. A large-scale, untargeted proteomic analysis revealed for the first time that all three—ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2—can modulate the abundance of 103 of the 7619 identified proteins, including proteins related to spermiogenesis and immunity, as well as others that are less well characterized. This report reinforces the importance of functionally characterizing GPA2 and GPB5 by showing the first results toward the characterization of their spermatogenesis related function in a specie of evolutive interest, the small-spotted catshark. In the future, a complete characterization of GPA2 and GPB5 will further our understanding of the functional evolution of glycoprotein hormones—a keystone group of hormones for vertebrate physiology and critical targets for fertility modulation in both livestock and humans.

## 1. Introduction

GPA and GPB belong to the glycoprotein hormone (GPH) family and have been identified in almost all non-vertebrate bilaterians [1]. Glycoprotein hormones diversification occurred at the root of gnathos- tomes *via* two whole-genome duplications (1R and 2R), which generated the GPH α- and β-subunits, as well as GPA2 and GPB5. The α-subunit forms heterodimers with the βFSH, βLH or βTSH subunits to produce the

gonadotropin hormones, FSH and LH, and the thyrotropin hormone, TSH. These hormones are involved in the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroid (HPT) axes by activating their respective G-protein-coupled receptors (FSHR, LHR and TSHR), as observed in vertebrates ranging from chondrichthyans (sharks, rays and chimeras) to mammals [2–4].

GPA2 and GPB5 are the homologues of the non-vertebrate GPA and GPB proteins [5–7]. Based on the heterodimeric bioactive hormones

<footnote><sup>a</sup> Université de Caen Normandie, Marine Ecosystems and Organisms Research lab (MERSEA), UR 7482, 14032 Caen, cedex 5, France</footnote>

<footnote><sup>b</sup> Université de Caen Normandie - Plateforme PROTEOGEN, US EMerode, 14032 Caen, cedex 5, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: pascal.sourdaine@unicaen.fr (P. Sourdaine).</footnote>
FSH, LH and TSH, the single-chain GPB5#GPA2 constructs were pro-
duced for human and rat proteins and they were able to activate TSHR,
but not FSHR or LHR [8,9]. However, several findings do not support the
formation of a GPA2-GPB5 heterodimer under physiological conditions.
These include the absence of the stabilizing seatbelt region in GPB5, the
presence of GPA2 and GPB5 in different cell types, systemic blood
concentrations below the dimerization Kd, and the fact that the heter-
odimer has never been identified under these conditions [6,10,11].
These findings have led to the hypothesis that GPA2 and GPB5 might be
bioactive as monomers and/or homodimers and act as paracrine and/or
autocrine factors [7,10].

Previous reports suggest that GPA2 and GPB5 are involved in regu-
lating thyroxine production [8,9], bone formation [12], the immune
response [13] and ovary function [14] in mammals. However, deleting
either the *gbp5* or *gpa2* gene in mice did not produce an obvious
phenotype [15]. Since a double knockout of both *gpa2* and *gbp5* has not
yet been performed, it cannot be excluded that GPA2 and GPB5 exert
similar functions and can rescue each other. In protostomes, GPA2 and
GPB5 also have pleiotropic functions. With regard to male reproduction,
RNA interference targeting the receptor of GPA2 and GPB5 resulted in
an inability to maintain a germline stem cell niche in the nematode
*Caenorhabditis elegans* [16] and disrupted spermiogenesis in the mos-
quito *Aedes aegypti* [17]. Because the physiological roles of GPA2/GPB5
remain poorly documented, further studies are needed to identify the
structure of physiologically bioactive hormones and better understand
how GPA2 and GPB5 regulate biological processes, including
gametogenesis.

In the catshark *Sclyliorhinus canicula*, a functional approach under *in vitro* conditions revealed that recombinant ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2 were able to activate all three ScFSHR, ScLHR and ScTSHR [7]. This contrasts with the results obtained in the *Cal*- *lorhinchus milii* chimera, in which highly specific receptor-hormone interactions were observed [3]. Expression analyses in *S. canicula* showed *gpa2* expression in male germ cells, including elongated spermatids, and the coexpression of *gbp5*, *tshr*, *fshr* and *lhr* in Sertoli cells. The highest levels of *gbp5* and *tshr* mRNAs were associated with the testicular zone at the stage of spermiogenesis [4,7]. These results suggest that GPA2 and GPB5 have paracrine and/or autocrine functions in regulating catshark spermiogenesis. The catshark is therefore an interesting model for studying the evolution and coexistence of putative ancestral regulatory pathways, which are potentially mediated by GPA2 and GPB5, and the emergence of the pituitary hormone-mediated regulation in the chon- drichthyan clade.

In this context, the present study aimed to explore the effects of re-
combinant ScGPA2, ScGPB5 and single-chain ScGPB5#ScGPA2 proteins
on testicular explants at stages of spermiogenesis in the catshark
*S. canicula* by using a proteomic approach to gain a first understanding.

**2. Materials and methods**

2.1. *Animals and tissue sampling*

Adult male catsharks (body weight: 873 ± 115 g; total length: 64 ± 3 cm) were provided by the Marine Biological Station of Roscoff (Centre de Ressources Biologiques – CRB Sorbonne Université-EMBRC, France). Animals were acclimated for a minimum of two weeks prior to sample collection. Catsharks were killed through MS222 overdose immersion following European Directive 2010/63/UE guidelines for animal welfare. Testicular tissue was harvested and cross-sectioned, then immediately placed in ice-cold Gautron's buffer (pH 7.8, 890 mosmol kg-1) [18] containing 58 mM trimethylamine-N-oxide (TMAO, Sigma, 317594). Tissue sections from 4 specimens were microdissected to isolate the zone of cysts with spermatids (stages XIII to XVIII) [19] and preserved in Gautron-TMAO buffer supplemented with 60 mg/ml penicillin, 100 mg/ml streptomycin and 250 µg/ml amphotericin, until explant culture.

2.2. *Production of recombinant independent subunits, ScGPA2 and ScGPB5, and single-chain ScGPB5#ScGPA2*

Recombinant hormone production and characterization followed established protocols Jeanne et al., 2025 [7]. Briefly, GenScript Biotech (Rijswijk, Netherlands) synthesized pTarget expression vectors encoding *S. canicula* sequences for *gpa2* (*Sc-gpa2*), *gbp5* (*Sc-gbp5*), and the chimeric construct *gbp5#gpa2* (*Sc-gbp5#Sc-gpa2*). HEK293T cells (80% of confluence in 75 cm² flasks) were transfected with these constructs using FuGENE HD (Promega) following manufacturer specifications. Supernatants from one large batch culture (3.6 ml from one 75 cm2 flask for each recombinant hormone) were harvested after 24 h incubation and concentrated *via* Amicon® 3 K filtration units (Millipore). Control preparations utilized HEK293T cells transfected with empty pTarget vectors. Recombinant protein quantification employed high-resolution NanoLC-ESI-MS/MS proteomic analysis with internal standards consisting of synthetic peptides derived from tryptic digestion of *ScGPA2* (EEIEIFTAK) and *ScGPB5* (EFTFLAK), supplied by CliniSciences (Nanterre, France). Biological activity predictions were calculated using previously established sigmoidal dose-response relationships for ScTSHR, *ScFSHR* and *ScLHR* receptors [7].

2.3. *In vitro incubation of S. canicula testis explants at stages of spermiogenesis*

Microdissected testicular regions were sectioned into 1mm³ explants and cultured in groups of 10 within 450 µl culture medium composed of L-15, 20 mM Hepes, 0.5% bovine serum albumin, 1% FBS, 0.25% heat-inactivated *S. canicula* serum, 4.6 mM D(+) glucose, 280 mM NaCl, 330 mM urea, 58 mM TMAO, and antimicrobials (60 mg/ml penicillin, 100 mg/ml streptomycin, 250 µg/ml amphotericin). Three independent explant groups (n = 3) received 50 µl treatments of conditioned media from transfected HEK293T cells. Due to the requirement for immediate use of recombinant proteins to maintain bioactivity, hormone concentrations were determined retrospectively: ScGPA2 (5.50 nM), ScGPB5 (2.71 nM), or single-chain ScGPB5#ScGPA2 (0.03 nM). Control explants received conditioned media from empty vector-transfected HEK293 cells. Following previous optimization studies [20–23], incubations proceeded for 22 h at 17 °C. Explants and media were subsequently collected and stored separately at –80 °C.

2.4. *Protein extraction*

Protein extraction was conducted on testicular explants following treatment with *ScGPA2* (5.50 nM), *ScGPB5* (2.71 nM), single-chain *ScGPB5#ScGPA2* (0.03 nM), or control media, with independent triplicates per condition. Frozen specimens were homogenized using a T25 digital Ultra-Turrax® in 2.5 volumes of ice-cold extraction buffer containing 6 M urea, 2 M thiourea, 40 mM Tris-Base, 2% CHAPS, 1 mM EDTA, 0.5 mM DTT, 1 mM AEBSF, and 10 µM E64 protease inhibitors. Following sonication and 1 h incubation on ice, samples were centrifuged at 15,000g for 30 min at 4 °C, then ultracentrifuged at 105,000 g for 1 h at 4 °C. Protein quantification of final supernatants was performed using bicinchoninic acid assay (Sigma-Aldrich, Merck, France).

2.5. *NanoLC-ESI-MS/MS analysis*

Protein samples (5 µg each) underwent processing *via* a modified gel-
aided sample preparation (GASP) methodology [24], employing gel
plugs sectioned into two fragments prior to overnight enzymatic diges-
tion at 37 °C using a trypsin/lysine-C mixture (400 ng per sample; V5073
Promega). Peptide purification and concentration were achieved using
C18 microcolumns (Omix C18 10 µl, Agilent), followed by chromato-
graphic separation on a NanoElute ultrahigh-pressure nanoflow system
(nanoLC, Bruker Daltonics). The separation setup included a C18 pep-
map 100 precolumn (5 mm × 300 µm i.d., Thermo Scientific) and a
temperature-controlled (50 °C) reversed-phase Reprosil analytical col-
umn (25 cm × 75 µm i.d.) containing 1.6 µm C18-coated porous silica
particles (Ionopticks). Chromatographic separation employed binary
mobile phases: 0.1% formic acid in water (phase A) and 0.1% formic
acid in acetonitrile (phase B). A 250 nl/min flow rate was maintained
with a gradient elution starting at 2% phase B, increasing to 30% over
70 min, rising to 37% within 5 min, followed by an 85% phase B wash
cycle and column re-equilibration. Mass spectrometric detection utilized
a TIMS-TOF pro instrument (Bruker Daltonics) equipped with a modi-
fied nano electrospray source (CaptiveSpray, Bruker Daltonics). Ioni-
zation parameters included 1400 V spray voltage and 180 °C capillary
temperature. Spectral acquisition occurred in positive ion mode span-
ning 100–1700 m/z with an ion mobility range of 0.60–1.60 1/k0. The
instrument operated in PASEF DIA mode excluding singly charged
species, utilizing 16 variable isolation windows across 400–1200 m/z.

2.6. *Database searching*

Protein identification and label-free quantification (LFQ) utilizing extracted ion chromatograms (XIC) were executed through DIA-NN software (version 1.8.2) (https://www.nature.com/articles/s41592-019-0638-x). The *S. canicula* predicted proteome from NCBI served as the reference database for library-free searching and spectral library construction. Retention time prediction and mass extraction accuracy employed default settings (parameter 0.0), enabling automatic mass and retention time calibration within DIA-NN. Peptide identification and quantification relied on the six highest-intensity fragments ranked by spectral library intensities. Permitted variable modifications included N-terminal acetylation and methionine oxidation, while cysteine propionamide modification was designated as fixed. Trypsin/P specificity was applied during database searching. Quality filtering maintained a 1% false discovery rate at the peptide level, and retention time-dependent

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Quality control and differentially abundant proteins (DAPs) in *S. canicula* testicular explants, at stages of spermiogenesis, treated with recombinant *ScGPA2* 5.50 nM, *ScGPB5* 2.71 nM and single-chain *ScGPB5#ScGPA2* 0.03 nM. A. A principal component analysis (PCA) on biological replicates (n = 3) of each condition corresponding to control, *ScGPA2* 5.50 nM, *ScGPB5* 2.71 nM and *ScGPB5#ScGPA2* 0.03 nM treatments, considering the 500 most variable catshark proteins. The black circles represent the clusters obtained using k-means clustering with a k parameter of four. The Cos2 color gradient indicates the samples' quality representation, considering both the selected dimensions. B. A venn diagram mapping the 933 differentially abundant proteins (DAPs) after all three treatments, highlighted by an ANOVA test. C. Heatmaps representing Log2ratio cluster abundance profiles, which were identified using k-means clustering with a k parameter of four for each treatment, taken independently. D. The means of DAPs Log2ratio by condition and by cluster were calculated and statistical analysis was performed using Tukey's Honest Significant Difference (HSD) test with P-value <0.05. C1: cluster 1; C2: cluster 2; C3: cluster 3; C4: cluster 4.</figcaption></figure>
cross-run normalization was implemented. Database interrogation yielded 7619 protein identifications across all experimental replicates and conditions.

## 2.7. Sample quality control

Protein abundance values for the 7619 identified species were transformed to Log2 ratios by dividing individual sample abundances by corresponding control group means. Principal component analysis (PCA) was conducted using the 500 most variable proteins as input matrix through the FactomineR package [25] in RStudio software (https://www.rstudio.com/). Subsequent k-means clustering analysis employed the Factoextra package [26] with k = 4 parameter settings, enabling replicate assignment to corresponding clusters (Fig. 1A).

## 2.8. Bioinformatics

Following Jeanne et al., 2023 [27] methodology and considering limited *S. canicula* protein annotation resources, the 7619 identified proteins underwent homology searching against the *Mus musculus* proteome (Proteome ID: UP000000589) obtained from UniProt (https://www.uniprot.org/) to leverage comprehensive annotation databases. BLASTp analysis was executed via the Galaxy platform (https://usegalaxy.org/) with stringent parameters: minimum 40% identity and coverage, maximum e-value of $1e^{-05}$. Differential abundance analysis of DIA-NN output employed the DEP package [28] in RStudio. Protein filtering retained species detected in minimum 2 of 3 replicates within at least one experimental condition. Missing values were imputed using PERSEUS normal distribution modelling, while differential expression analysis utilized linear modelling with empirical Bayes statistics. Significance thresholds required minimum 1.2-fold abundance changes ($Log2(1.2) = 0.27$) and P-values $\le 0.05$ ($-Log10(0.05) = 1.3$) determined through PERSEUS ANOVA testing. Triplicate Log2 ratios were averaged per treatment condition (ScGPA2 5.50 nM, ScGPB5 2.71 nM, ScGPB5#ScGPA2 0.03 nM, control). Analysis identified 933 differentially abundant proteins (DAPs) relative to controls across treatment conditions. Treatment-specific protein mapping utilized the ggvenn package in RStudio [29], demonstrating that 21% of DAPs responded to multiple treatments while 79% showed treatment-specific responses. Independent k-means clustering ($k = 4$) was performed for DAPs within each treatment group, generating four clusters per treatment. Clusters encompassed highly increased (C1) and decreased (C4) proteins, alongside moderately increased (C2) or decreased (C3) abundance groups. Heatmap visualization displayed Log2 ratio variations across clusters and treatments. Statistical evaluation of treatment-induced protein level changes employed cluster and treatment-averaged Log2 ratios analyzed via Tukey's Honest Significant Difference (HSD) testing ($P < 0.05$). To evaluate consistency of highly modified DAPs across treatments despite potentially non-significant individual changes, standard error of the mean (SEM) calculations were performed for DAPs in clusters 1 (C1.A2, C1.B5, C1.B5A2) or 4 (C4.A2, C4.B5, C4.B5A2). SEM values were expressed as percentages of corresponding treatment means, with SEM $\le 33\%$ defining DAPs exhibiting consistent Log2 ratio patterns across treatments.

## 2.9. Functional annotation

Initially, gene ontology-based functional annotation employed ClueGO software [30] for mouse ortholog accessions within clusters 1 (C1.A2, C1.B5, C1.B5A2) and 4 (C4.A2, C4.B5, C4.B5A2), applying parameters consistent with Jeanne et al., 2025 [21]. However, limited *M. musculus* annotations for the identified DAPs yielded minimal enriched terms with few associated proteins (data not presented). Next, additional annotation resources (eggNOG Mapper) and frameworks (g: profiler; clusterProfiler with over-representation analysis (ORA); clusterProfiler with gene set enrichment analysis (GSEA)) were employed

without obtaining more relevant results. Consequently, functional characterization and spermatogenesis relevance assessment for C1 and C4 DEPs utilized comprehensive NCBI database mining and literature review approaches. Manual functional classification established protein categories based on defined criteria: Spermatids, Sperm motility, Infer- tility/subfertility, Spermatogenesis or oogenesis, Testicular function, Immunity, Disease, Other, Uncharacterized.

# 3. Results

## 3.1. Theoretical efficiencies of ScGPA2, ScGPB5, and ScGPB5#ScGPA2 on ScTSHR, ScFSHR and ScLHR

The concentrations used to treat the testicular explants were of 5.50 nM for ScGPA2, 2.71 nM for ScGPB5, and 0.03 nM for ScGPB5#ScGPA2. This heterogeneity in concentrations is explained by the direct use of the conditioned media containing the recombinant hormone in order to avoid the freezing step, which decreases its efficiency. The absolute concentrations were identified afterward by NanoLC-ESI-MS/MS. Based on the sigmoidal dose-response equations for the catshark ScTSHR, ScFSHR, and ScLHR [7], 2.71 nM of ScGPB5 appeared to be the most efficient treatment because it activated all three ScGPHRs at 100% efficiency. Next was 0.03 nM of single-chain ScGPB5#ScGPA2, which activated all three ScGPHRs with an average efficiency of 59%. At 5.50 nM, ScGPA2 activated ScTSHR, ScLHR and ScFSHR with an efficiency of 80%, 51% and 25%, respectively (Table 1).

## 3.2. Effects of treatment with ScGPA2, ScGPB5 or ScGPB5#ScGPA2 on the proteome of *S. canicula* testicular explants at stages of spermiogenesis

Proteomic analyses of testicular treated explants allowed the identification of a total of 7619 proteins. To verify the homogeneity of replicates across the different conditions, the 500 most variable proteins were analyzed using Principal Component Analysis (PCA) followed by k-means clustering, which revealed that replicates were grouped according to treatment (Fig. 1A). The ANOVA revealed that 212, 449 and 525 differentially abundant proteins (DAPs) were associated to ScGPA2, ScGPB5 and ScGPB5#ScGPA2 treatment, respectively (Fig. 1B). Protein mapping revealed that 79% of DAPs were associated with one treatment, including 11% (99 DAPs) associated to ScGPA2, 31% (285 DAPs) associated to ScGPB5, and 37% (342 DAPs) associated to ScGPB5#ScGPA2 (Fig. 1B). Only 18% and 5% of DAPs were common to two and three treatments, respectively. A Log2ratio analysis of the DAPs was therefore carried out treatment-by-treatment using the k-clustering method ($k = 4$) (Fig. 1C). The clusters C1 (C1.A2, C1.B5, C1.B5A2) and C4 (C4.A2, C4.B5, C4.B5A2) contained a total of 103 unique accessions (Fig. 1C), 16 of which were common to two or three treatments. These clusters appeared highly sensitive to all three treatments, with mean Log2ratio of $2.47 \pm 0.15$ and $-2.37 \pm 0.19$ for C1 and C4, respectively (Fig. 1D). The clusters C2 (C2.A2, C2.B5, C2.B5A2) and C3 (C3.A2, C3.B5, C3.B5A2) appeared less sensitive to treatments, with a mean Log2ratio of $0.73 \pm 0.03$ (C2) and $0.63 \pm 0.01$ (C3) (Fig. 1D). The k-clustering analysis per treatment showed that: the ScGPA2 treatment resulted in the strong increase in abundance of 10 DAPs (C1.A2) and the high decrease in abundance of 7 DAPs (C4.A2); the ScGPB5 treatment induced high

<table><caption>**Table 1**

Theoretical efficiencies of recombinant ScGPA2, ScGPB5 and single-chain ScGPB5#ScGPA2 on ScTSHR, ScFSHR and ScLHR.</caption><thead><tr><th></th><th colspan="3">Theoretical efficiency (%)</th></tr><tr><th></th><th>ScGPA2</th><th>ScGPB5</th><th>ScGPB5#ScGPA2</th></tr></thead><tbody><tr><td></td><td>5.50 nM</td><td>2.71 nM</td><td>0.03 nM</td></tr><tr><td>ScFSHR</td><td>25</td><td>100</td><td>62</td></tr><tr><td>ScLHR</td><td>51</td><td>100</td><td>60</td></tr><tr><td>ScTSHR</td><td>80</td><td>94</td><td>55</td></tr></tbody></table>
increase in abundance of 34 DAPs (C1-B5) and high decrease in abundance of 32 DAPs (C4-B5); the treatment with ScGPB5#ScGPA2 induced the high increase in abundance of 29 DAPs (C1.B5A2) as well as high decrease in abundance of 15 DAPs (C4.B5A2) (Fig. 1C). To explore the similar and specific effects of treatments on testicular explant proteomes, the distribution of the 103 highly modulated DAPs was constructed based on the calculation of variance in abundance between treatments (Fig. 2A). In total, 73 out of the 103 DAPs exhibited a SEM < 33% (Fig. 2A), underlining the similar effect of the three treatments. The 10 DAPs presenting the lowest SEM were associated to the three treatments and included the proteins whose abundance increased such as IFITM3 with a Log₂ratio of 2.44 to 2.92, LRRC69 with a Log₂ratio of 3.13 to 3.85, BTBD2 with a Log₂ratio of 1.66 to 2.07, TM4SF4 with a Log₂ratio of 1.74 to 2.13, SLC2A1 with a Log₂ratio of 1.56 to 1.98, TMEM9B with a Log₂ratio of 1.37 to 1.97, GANC with a Log₂ratio of 1.34 to 2, RAB39B with a Log₂ratio of 0.96 to 1.41, and C1GALT1 with a Log₂ratio of 1.23 to 1.79, while abundance of TMEM164 was decreased with a Log₂ratio of -3.69 to -5.48 (Fig. 2B). In the opposite, of the 10 DAPs with the highest SEM, which highlights the preferential effect of one of the three treatments, 9 were associated with the ScGPB5 treatment. These included XP_038639919 with a Log₂ratio of 1.84, ARFGAP1 with a Log₂ratio of -1.36, HEXD with a Log₂ratio of -1.62, IRGC with a Log₂ratio of -2.46, RNF185 with a Log₂ratio of -1.46, GPHB5 with a Log₂ratio of 2.62, ABCB9 with a Log₂ratio of -1.34, XP_038663965 with a Log₂ratio of 1.93, and XP_038633100 with a Log₂ratio of -1.5. The tenth DAP was AKAP17A, which was associated with the ScGPB5#ScGPA2 treatment with a Log₂ratio of -1.97 (Fig. 2C).

Functional annotation of the 103 highly modulated DAPs, selected on the basis of a strong and statistically significant modulation in response to at least one treatment, was carried out manually using the NCBI database and the literature in order to highlight their functions and potential significance during spermatogenesis in other species. Based on the information obtained, the 103 highly modulated DAPs were categorized under the following keywords: "Spermatids" (10 DAPs), "Sperm motility" (10 DAPs), "Infertility/Subfertility" (20 DAPs), "Spermatogenesis or oogenesis" (7 DAPs), "Testicular function" (9 DAPs), "Immunity" (6 DAPs), "Disease" (11 DAPs), "other" (24 DAPs), and "Uncharacterized" (6 DAPs) (Table 2). Except for the keywords "Infertility/Subfertility", which was mainly associated with the ScGPB5#ScGPA2 treatment, and "Testicular function", which was mainly associated with the ScGPA2 treatment, all keywords were mainly associated with the ScGPB5 treatment (Fig. 2D).

The following proteins were highlighted among the most highly increased (Clusters C1.A2, C1.B5, and C1.B5A2) or decreased (Clusters C4.A2, C4.B5, and C4.B5A2) DAPs (Fig. 1C) (Table 2). With the ScGPA2 treatment, the 10 most significantly DAPs whose abundance increased were: AGR3; LRRC69; IFITM3; RIC1; GANC; SLC2A1; IFTAP; TM4SF4; GOLGA4; BTBD2; with a Log₂ratio ranging from 4.27 to 1.66 (Table 2). The ScGPA2 treatment also resulted in the strong decrease in the abundance of CHTF18, TMEM164, BTD, TMEM196, TRIM69, ACTN2, IGKV4-55, and XP_038673256 with a Log₂ratio ranging from -3.84 to -1.73. The ScGPB5 treatment induced a high increase in the abundance of COL6A3, LRRC69, XP_038663118, SPINK2, ZDHHC23, XP_038643196, GPHB5, RIPOR3, NPHS1, and IFITM3, with a Log₂ratio

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Identification of highly modulated DAPs that are regulated similarly across all three treatment or preferentially by a single treatment, followed by their manual functional annotation. A. Distribution of the standard error of the mean (SEM), expressed as a percentage of the mean across the three treatments, calculated with the Log₂ratio for each of the 103 DAPs identified in at least one of the clusters 1 (C1.A2, C1.B5, C1.B5A2) or 4 (C4.A2, C4.B5, C4.B5A2). The 33% is indicated by the red line. B. Log₂ratio of the ten most homogenous highly modulated DAPs across the three treatments. C. Log₂ratio of the ten most heterogenous highly modulated DAPs across the three treatments. D. Manual functional annotation of the 103 most modulated DAPs, presented with the percentage of DAPs per treatment and per keyword, with 100% corresponding to the total number of DAPs associated with each keyword. C1: cluster 1; C4: cluster 4. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<caption>**Table 2**</caption>

Functional annotation of the highly differentially abundant proteins (DAPs) highlighted in clusters C1 and C4 (in bold) of testicular *S. canicula* explants, at stages of spermiogenesis, treated with ScGPA2 5.50 nM, ScGPB5 2.71 nM or ScGPB5#ScGPA2 0.03 nM.

<table><thead><tr><th rowspan="2">Keyword</th><th rowspan="2">DAPs</th><th rowspan="2">Full name</th><th rowspan="2">Function</th><th rowspan="2">Potential significance in spermatogenesis (if any)</th><th rowspan="2">Species</th><th colspan="3">Log2(ratio)</th><th rowspan="2">Reference</th></tr><tr><th>ScGPA2</th><th>ScGPB5</th><th>ScGPB5-ScGPA2</th></tr></thead><tbody><tr><td rowspan="15">Spermatids</td><td>ACTB</td><td>Actin, cytoplasmic 1</td><td>Cytoskeleton</td><td>Supports transport of spermatids and residual bodies/phagosomes during spermatogenesis.</td><td>Rat</td><td>0.97</td><td>1.74</td><td>2.68</td><td>[74,75]</td></tr><tr><td>ATP5MK (USMG5)</td><td>ATP synthase membrane subunit k</td><td>ATP synthesis</td><td>Related to diaphorase activity in sperm.</td><td>Mice</td><td>1.37</td><td>1.79</td><td>2.38</td><td>[33]</td></tr><tr><td>CAPN3</td><td>Calpain-3</td><td>Calcium-dependent endopeptidase</td><td>Deletion of CAPN3 had no effect on fertility.</td><td>Cattleyak Mice</td><td>-0.21</td><td>1.97</td><td>3.4</td><td>[76,77]</td></tr><tr><td>H2AX</td><td>Histone H2AX</td><td>Nucleosome structure</td><td>Downregulated in spermatid-deficient testis.</td><td>Mice</td><td>0.13</td><td>1.58</td><td>1.44</td><td>[78]</td></tr><tr><td>KIF16B</td><td>Kinesin-like protein KIF16B</td><td>Intracellular trafficking</td><td>Involved in the histone-to-protamine transition.</td><td>Lobster</td><td>0.01</td><td>1.72</td><td>2.53</td><td>[44]</td></tr><tr><td>SPCS3</td><td>Signal peptidase complex subunit 3</td><td>Signal peptide processing</td><td>Involved in nuclear shaping and acrosome formation.</td><td>Sheep</td><td>1.53</td><td>1.83</td><td>4.25</td><td>[39]</td></tr><tr><td>ARFGAP1</td><td>ADP-ribosylation factor GTPase activating protein 1</td><td>GTPase activator</td><td>Related to sperm acrosomal and plasma membranes.</td><td>Boar</td><td>0.17</td><td>-1.36</td><td>0.49</td><td>[79]</td></tr><tr><td>CLCN7 (CLC7)</td><td>H(+)/Cl(-) exchange transporter 7</td><td>Chloride transporter</td><td>Regulate the Golgi-ER membrane traffic with ARF1 and may influence hyperactive motility in ejaculates.</td><td>Human Mice</td><td>0.15</td><td>-1.48</td><td>-0.17</td><td>[59]</td></tr><tr><td>MRPS17 (RPMS17)</td><td>Small ribosomal subunit protein uS17m</td><td>Mitochondrial ribosomal proteins</td><td>CLCs might be involved in sperm capacitation.</td><td>Primates</td><td>-0.41</td><td>-1.44</td><td>-1.03</td><td>[80]</td></tr><tr><td>MS4A15</td><td>Membrane-spanning 4-domains subfamily A member 15</td><td>Cell surface receptor signaling pathway</td><td>MS4A15 is an oncogene in ovarian cancer. MS4A proteins may form oligomers in sperm cell membranes, which could affect fertility.</td><td>Mouse Human</td><td>-0.72</td><td>-1.44</td><td>-1.15</td><td>[52,81]</td></tr><tr><td>CRYAB</td><td>Alpha-crystallin B chain</td><td>Molecular chaperone holding proteins in soluble aggregates</td><td>CRYAB is upregulated in testis with high sperm motility.</td><td>Chicken Human</td><td>0.48</td><td>1.45</td><td>-0.08</td><td>[47,48]</td></tr><tr><td>GANC</td><td>Glucosidase alpha, neutral C</td><td>Glucose release</td><td>Glucose is an energy source required for sperm motility.</td><td>Human</td><td>2</td><td>1.44</td><td>1.34</td><td>[36,37]</td></tr><tr><td>IFITM3</td><td>Interferon induced transmembrane protein 3</td><td>Antiviral protein</td><td>Ifitm-1 may enhance the motility of spermatozoa.</td><td>Mice</td><td>2.92</td><td>2.44</td><td>2.64</td><td>[35]</td></tr><tr><td>PPP3CA</td><td>Protein phosphatase 3 catalytic subunit alpha</td><td>Signaling pathways</td><td>PPP3CA is involved in a pathway crucial for sperm motility.</td><td>chicken</td><td>1.34</td><td>1.13</td><td>1.95</td><td>[82]</td></tr><tr><td rowspan="6">Sperm motility</td><td>ACTN2</td><td>Alpha-actinin-2</td><td>Cytoskeleton</td><td>Downregulation of actn2 gene was associated with low sperm motility.</td><td>Rooster</td><td>-1.98</td><td>-1.07</td><td>-1.43</td><td>[83]</td></tr><tr><td>CCDC38</td><td>Coiled-coil domain-containing protein 38</td><td>Ubiquitin-modified histone reader</td><td>CCDC38 is required for the biogenesis of sperm flagellum.</td><td>Mice</td><td>-1.36</td><td>-1.92</td><td>-0.94</td><td>[56]</td></tr><tr><td>FILIP1</td><td>Filamin-A-interacting protein 1</td><td>Promotes the degradation of filamin A</td><td>Filamin A plays a role in endocytosis of tight junction at the BTB and possibly in axoneme formation of sperm.</td><td>Human Mice</td><td>-0.13</td><td>-2.07</td><td>0.11</td><td>[57]</td></tr><tr><td>IRGC (IRGC1)</td><td>Immunity related GTPase cinema</td><td>GTPase activity</td><td>Fibrous sheath integrity and sperm motility.</td><td>Mice</td><td>0.3</td><td>-2.46</td><td>-0.78</td><td>[58]</td></tr><tr><td>RYBP</td><td>RING1 and YY1-binding protein</td><td>DNA binding activity and</td><td>Rybp orchestrates spermatogenesis via</td><td>Mice</td><td>-0.5</td><td>-1.48</td><td>0.34</td><td>[60]</td></tr></tbody></table>

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<table><caption>**Table 2 (continued)**</caption><thead><tr><th rowspan="2">Keyword</th><th rowspan="2">DAPs</th><th rowspan="2">Full name</th><th rowspan="2">Function</th><th rowspan="2">Potential significance in spermatogenesis (if any)</th><th rowspan="2">Species</th><th colspan="3">Log2(ratio)</th><th rowspan="2">Reference</th></tr><tr><th>ScGPA2</th><th>ScGPB5</th><th>ScGPB5-ScGPA2</th></tr></thead><tbody><tr><td rowspan="10">Infertility Subfertility</td><td>SLC25A28 (MFRN2)</td><td>Mitoferrin-2</td><td>transcription corepressor activity</td><td>regulating meiosis and sperm motility. MFRN2-knockout mice showed a reduction in sperm number and motility. Cox7b2 KO mice produced poorly motile infertile spermatozoa.</td><td>Mice</td><td>-0.99</td><td>-1.31</td><td>-4.45</td><td>[55,84]</td></tr><tr><td>COX7B</td><td>Cytochrome c oxidase subunit 7B, mitochondrial</td><td>Part of respiratory chain complex IV</td><td>Mutation of eEF1A1b resulted in abnormal spermiogenesis.</td><td>mice</td><td>2.11</td><td>2.45</td><td>4.2</td><td>[34]</td></tr><tr><td>EEF1A1</td><td>Eukaryotic translation elongation factor 1 alpha 1</td><td>Translation</td><td>Acts upstream Gmip/ RhoA to regulate cortical actin remodeling and spindle positioning during early mitosis. Lack of NWC reduced sperm competitiveness and its ability to fertilize.</td><td>Nile tilapia</td><td>1.08</td><td>0.59</td><td>2.09</td><td>[49]</td></tr><tr><td>GEM (KIR)</td><td>GTP binding protein overexpressed in skeletal muscle</td><td>Regulatory protein in receptor-mediated signal transduction</td><td>KCTD19 is testis-specific and is transcriptional regulation of meiotic genes. Its loss leads to spermatogenetic failure.</td><td>Human</td><td>1.71</td><td>1.55</td><td>3.82</td><td>[85]</td></tr><tr><td>IFTAP (NWC)</td><td>Intraflagellar transport associated protein</td><td>Intracellular transport particle A binding</td><td>LRRC69 significantly overrepresented in patients with spermatogenic failure. Low and irregular abundance in patients with hypospermatogenesis and Sertoli cell-only syndrome.</td><td>Mice</td><td>1.9</td><td>1.86</td><td>1.04</td><td>[86]</td></tr><tr><td>KCTD12</td><td>Potassium channel tetramerisation domain containing 12</td><td>Regulation of G protein-coupled neurotransmitter receptor</td><td>Upregulated in testis with reproductive dysfunction. Reduced abundance associated with reproductive impairment.</td><td>Human Mice</td><td>0.45</td><td>0.72</td><td>1.92</td><td>[87,88]</td></tr><tr><td>LRRC69</td><td>Leucine-rich repeat-containing protein 69</td><td>Intracellular signal transduction</td><td>Acrosin inhibitor involved in acrosome protection. Spink2 KO mice have azoospermia. XPC mutant mouse have no significant reproductive defect while disruption of mHR23B (XPC partner in NER) causes defective spermatogenesis.</td><td>Human</td><td>3.65</td><td>3.13</td><td>3.85</td><td>[50]</td></tr><tr><td>NPHS1</td><td>Nephrin</td><td>Cell adhesion molecule</td><td>Altered levels may be associated with male infertility.</td><td>Human</td><td>1.46</td><td>2.45</td><td>4</td><td>[51]</td></tr><tr><td>PTPN6</td><td>Protein tyrosine phosphatase non-receptor type 6</td><td>Signaling molecules</td><td>Chtf18-null males showed oligospermia and subfertility.</td><td>Human Mice</td><td>0.15</td><td>1.42</td><td>1.09</td><td>[89,90]</td></tr><tr><td>SLC2A1 (GLUT1)</td><td>Solute carrier family 2 member 1</td><td>Glucose transporter</td><td>Potential marker of asthenozoospermia.</td><td>Mice Rat</td><td>1.95</td><td>1.98</td><td>1.56</td><td>[38]</td></tr><tr><td>SPINK2</td><td>Serine peptidase inhibitor Kazal-type 2</td><td>Serine protease inhibitor</td><td>Regulates Vitamin B12 homeostasis, which</td><td>Mice</td><td>2.02</td><td>2.99</td><td>4.38</td><td>[40]</td></tr><tr><td>XPC (P125)</td><td>XPC complex subunit, DNA damage recognition and repair factor</td><td>Key component of the XPC complex involved in nucleotide excision repair</td><td>Regulates Vitamin B12 homeostasis, which</td><td>Mice</td><td>1.19</td><td>2.33</td><td>3.52</td><td>[45]</td></tr><tr><td>CASR</td><td>Calcium-sensing receptor</td><td>Calcium homeostasis</td><td>Altered levels may be associated with male infertility.</td><td>Human</td><td>-0.32</td><td>-0.98</td><td>-1.98</td><td>[91]</td></tr><tr><td>CHTF18</td><td>Chromosome transmission fidelity factor 18</td><td>Component of the replication factor C complex</td><td>Regulates Vitamin B12 homeostasis, which</td><td>Mice</td><td>-3.84</td><td>-1.71</td><td>-1.82</td><td>[65]</td></tr><tr><td>ESF1</td><td>ESF1 nucleolar pre-rRNA processing protein homolog</td><td>rRNA processing</td><td>Potential marker of asthenozoospermia.</td><td>Human</td><td>-0.92</td><td>-1.2</td><td>-0.74</td><td>[66]</td></tr><tr><td>FUT2</td><td>Fucosyltransferase 2</td><td>Galactoside 2-L-fucosyltransferase</td><td>Regulates Vitamin B12 homeostasis, which</td><td>Human Mice</td><td>-0.97</td><td>-0.7</td><td>-3.84</td><td>[63]</td></tr></tbody></table>

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<table><caption>**Table 2 (continued)**</caption><thead><tr><th rowspan="2">Keyword</th><th rowspan="2">DAPs</th><th rowspan="2">Full name</th><th rowspan="2">Function</th><th rowspan="2">Potential significance in spermatogenesis (if any)</th><th rowspan="2">Species</th><th colspan="3">Log2(ratio)</th><th rowspan="2">Reference</th></tr><tr><th>ScGPA2</th><th>ScGPB5</th><th>ScGPB5-ScGPA2</th></tr></thead><tbody><tr><td rowspan="10">Spermatogenesis or oogenesis</td><td>MRPS16 (RPMS16)</td><td>Mitochondrial ribosomal protein S16</td><td>Mitochondrial protein synthesis</td><td>is associated with semen quality.<br>MRPS16 may mitigate mitochondrial injury. Such injury can result in sperm defects.</td><td>C. elegans Mice</td><td>-0.75</td><td>-1.89</td><td>-0.91</td><td>[92,93]</td></tr><tr><td>NSUN6</td><td>NOP2/Sun RNA methyltransferase 6</td><td>Enables tRNA (C-5-)-methyltransferase activity</td><td>Mutations in NSUN7 are associated with male infertility.</td><td>Human Mice</td><td>-1.6</td><td>-1.48</td><td>-2.56</td><td>[67-69]</td></tr><tr><td>OSBPL6</td><td>Oxysterol binding protein like 6</td><td>Intracellular lipid receptors</td><td>OSBPL6 potentially related to semen volume. Mediate ATG5-dependent autophagosome formation during ferroptosis. Atg5 cKO mice are subfertile/ infertile.</td><td>Chicken</td><td>-1.44</td><td>-1.23</td><td>-2.24</td><td>[64]</td></tr><tr><td>TMEM164</td><td>Transmembrane protein 164</td><td>Positive regulator of ferroptosis</td><td>Potential marker of asthenozoospermia.</td><td>Human, Mice, Rat</td><td>-3.69</td><td>-4</td><td>-5.48</td><td>[54,94]</td></tr><tr><td>ZMYM4</td><td>Zinc finger MYM-type containing 4</td><td>Predicted to enable DNA binding activity</td><td>Involved in the double-strand break repair pathway. Possibly regulates gametogenesis, acrosome integrity, and fertilization.</td><td>Human</td><td>-1.24</td><td>-0.84</td><td>-2.74</td><td>[66]</td></tr><tr><td>BTBD2</td><td>BTB domain containing 2</td><td>Topoisomerase I binding protein</td><td>Male+F58:G99 lacking C1GALT1 related transferase (MGAT1) had defective spermatozoa and c1galt1 deletion had no effect on fertility.</td><td>Human</td><td>1.66</td><td>1.84</td><td>2.07</td><td>[46]</td></tr><tr><td>C1GALT1</td><td>Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyl-transferase 1</td><td>Transfer of Gal from UDP-Gal to GalNAc-alpha-1-R.</td><td>Ca2+ serves as a crucial second messenger in the modulation of various sperm physiological processes.</td><td>Mice</td><td>1.23</td><td>1.79</td><td>1.23</td><td>[95]</td></tr><tr><td>CACNG1</td><td>Calcium voltage-gated channel auxiliary subunit gamma 1</td><td>Voltage-gated calcium channel</td><td>Oocyte-derived thyristimulin and granulosa cell-expressed TSHR compose a paracrine system in the ovary. TMEM9B with CIC-3 or CIC-4 dramatically reduced chloride transporter activity in Xenopus oocytes and transfected HEK cells. Found in Sertoli cells.</td><td>Rat</td><td>0.56</td><td>1.99</td><td>1.13</td><td>[96]</td></tr><tr><td>GPHB5</td><td>Glycoprotein hormone beta 5</td><td>Cystine knot glycoprotein hormone</td><td>Pkia KO mice have no defects in testis weight, sperm number or sperm motility. Involved in radiation-induced spermatogonial stem cell apoptosis.</td><td>Rat</td><td>-0.15</td><td>2.62</td><td>-0.23</td><td>[14]</td></tr><tr><td>TMEM9B</td><td>Transmembrane protein 9B</td><td>Positive regulation of canonical NF-kappaB signal transduction</td><td>CLTA is involved in clathrin coat and modulate endocytosis in the testis.</td><td>Xenopus</td><td>1.37</td><td>1.91</td><td>1.97</td><td>[97]</td></tr><tr><td rowspan="4">Testicular function</td><td>PKIA</td><td>cAMP-dependent protein kinase inhibitor alpha</td><td>Inhibits C alpha and C beta catalytic subunits of the PKA</td><td>Involved in radiation-induced spermatogonial stem cell apoptosis.</td><td>Human Mice, Rat</td><td>-0.61</td><td>-1.34</td><td>-1.36</td><td>[98,99]</td></tr><tr><td>TRP53INP1</td><td>Transformation related protein 53 inducible nuclear protein 1</td><td>Antioxidant activity</td><td>CLTA is involved in clathrin coat and modulate endocytosis in the testis.</td><td>Mice</td><td>-0.4</td><td>-2.02</td><td>-1.15</td><td>[100]</td></tr><tr><td>CLTA</td><td>Clathrin light chain A</td><td>Main structural component of the lattice-type cytoplasmic face of coated pits and vesicles</td><td>Abundant in the testes of mice. Male knockouts are fertile.</td><td>Mice</td><td>1.32</td><td>1.29</td><td>2.21</td><td>[101]</td></tr><tr><td>GOLGA4</td><td>Golgin subfamily A member 4</td><td>Small GTPase binding activity</td><td>Abundant in the testes of mice. Male knockouts are fertile.</td><td>Mice</td><td>1.75</td><td>0.77</td><td>1.54</td><td>[102]</td></tr></tbody></table>

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<table><caption>**Table 2 (continued)**</caption><thead><tr><th rowspan="2">Keyword</th><th rowspan="2">DAPs</th><th rowspan="2">Full name</th><th rowspan="2">Function</th><th rowspan="2">Potential significance in spermatogenesis (if any)</th><th rowspan="2">Species</th><th colspan="3">Log2(ratio)</th><th rowspan="2">Reference</th></tr><tr><th>ScGPA2</th><th>ScGPB5</th><th>ScGPB5-ScGPA2</th></tr></thead><tbody><tr><td rowspan="12">Immunity</td><td>SLC7A1</td><td>Solute carrier family 7 member 1</td><td>Cationic amino acids transporter</td><td>L-arginine transport by Sertoli cells and peritubular cells.</td><td>Mice</td><td>1.42</td><td>2.42</td><td>3.45</td><td>[103]</td></tr><tr><td>WDR73</td><td>WD repeat domain 73</td><td>Assembly factor for the RNA cleavage module of Integrator</td><td>Abundant in the testes. WDR73 mutations in Galloway-Mowat syndrome.</td><td>Mice<br>Yellow</td><td>0.68</td><td>1.53</td><td>1.15</td><td>[104,105]</td></tr><tr><td>AKAP17A</td><td>A-kinase anchoring protein 17 A</td><td>Part of the spliceosome complex</td><td>Regulates pre-mRNA splicing and is found in the testis.</td><td>Human</td><td>0.62</td><td>0.44</td><td>-1.97</td><td>[61]</td></tr><tr><td>BTD</td><td>Biotinidase</td><td>Biocytin cleavage and biotinyl transferase</td><td>Biotin treatment increases cAMP levels, leading to PKA activation and testosterone production.</td><td>Mice</td><td>-2.81</td><td>-0.67</td><td>-0.68</td><td>[106]</td></tr><tr><td>PRKG1</td><td>Protein kinase cGMP-dependent 1</td><td>cGMP-dependent protein kinase</td><td>Increased in senescent Leydig cells. The testis specific RASEF is unessential for fertility and normal spermatogenesis.</td><td>Rat</td><td>-0.43</td><td>-1.34</td><td>-2.08</td><td>[107]</td></tr><tr><td>RASEF</td><td>RAS and EF-hand domain containing</td><td>Regulation of membrane traffic</td><td>Trim69 KO male mice are fertile, suggesting compensation by other TRIM family members. Suppresses the effects of RHO activity.</td><td>Mice</td><td>-0.85</td><td>-1.27</td><td>-2.13</td><td>[108]</td></tr><tr><td>TRIM69</td><td>Tripartite motif containing 69</td><td>ubiquitin-protein ligase</td><td>RIPOR3 is linked to immune cell infiltration.</td><td>Mice</td><td>-2.13</td><td>-0.38</td><td>0.16</td><td>[109]</td></tr><tr><td>RIPOR3</td><td>RIPOR family member 3</td><td>Bind to RHO protein</td><td>Negative regulation of viral genome replication.</td><td>Human</td><td>0.66</td><td>2.54</td><td>0.52</td><td>[110,111]</td></tr><tr><td>XP_038643196</td><td>Interferon-stimulated 20 kDa exonuclease-like</td><td>3'-5' exonuclease activity and RNA binding activity</td><td>Predicted to be involved in cellular defense response and regulation of complement activation.</td><td>Human<br>Mice</td><td>1.22</td><td>2.75</td><td>-0.21</td><td>NCBI<br>Gene</td></tr><tr><td>XP_038652419</td><td>Fucolectin-like</td><td>Fucose binding activity</td><td>H2-Aa expression is elevated in Leydig cells of the db/db mouse. H2-Aa loss led to MHC II deficiency in lc3b-/- mice. In the ovary, IGKV4-1 is upregulated after mating and downregulated before fertilization.</td><td>Xenopus</td><td>0.02</td><td>1.71</td><td>2.53</td><td>NCBI<br>Gene</td></tr><tr><td>H2-AA</td><td>histocompatibility 2, class II antigen A, alpha</td><td>Peptide antigen binding activity</td><td>Catalyze the K27-linked polyubiquitination of cGAS, thereby promoting its enzymatic activity in innate immune responses.</td><td>Mice</td><td>0.6</td><td>-2.28</td><td>-0.67</td><td>[112]</td></tr><tr><td>IGKV4-55</td><td>immunoglobulin kappa variable 4-55</td><td>Predicted to be part of immunoglobulin complex</td><td>Associated with differentiation and survival in ovarian cancer.</td><td>Black<br>rockfish</td><td>-1.82</td><td>-0.07</td><td>-0.16</td><td>[113]</td></tr><tr><td>RNF185</td><td>Ring finger protein 185</td><td>ubiquitin protein ligase activity</td><td>COL6A3 exosomes promote tumor dissemination and metastasis in epithelial ovarian cancer.</td><td>Human</td><td>0.53</td><td>-1.46</td><td>-0.38</td><td>[114,115]</td></tr><tr><td>AGR3</td><td>Anterior gradient 3, protein disulphide isomerase family member</td><td>Disulfide isomerase</td><td>EXTL3 produce the backbone structure of heparan sulfate (HS). The loss of HS leads to</td><td>Human</td><td>4.27</td><td>2.45</td><td>-0.54</td><td>[116]</td></tr><tr><td>Disease</td><td>COL6A3</td><td>Collagen type VI alpha 3 chain</td><td>One of the three alpha chains of type VI collagen</td><td>Human</td><td>0.39</td><td>3.33</td><td>0.24</td><td>[117]</td></tr><tr><td></td><td>EXTL3</td><td>Exostosin like glycosyltransferase 3</td><td>Glycosyltransferase</td><td>Drosophila</td><td>1.33</td><td>1.42</td><td>0.89</td><td>[118,119]</td></tr></tbody></table>

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<table><caption>**Table 2 (continued)**</caption><thead><tr><th rowspan="2">Keyword</th><th rowspan="2">DAPs</th><th rowspan="2">Full name</th><th rowspan="2">Function</th><th rowspan="2">Potential significance in spermatogenesis (if any)</th><th rowspan="2">Species</th><th colspan="3">Log2(ratio)</th><th rowspan="2">Reference</th></tr><tr><th>ScGPA2</th><th>ScGPB5</th><th>ScGPB5-ScGPA2</th></tr></thead><tbody><tr><td></td><td>RBM12B2</td><td>RNA-binding protein 12B-B</td><td>RNA binding activity</td><td>tumor-like germ cell growth.<br/>A potential modifier of human breast cancer risk and agressiveness. Associated with the progressive cone degeneration of Sturge-Weber Syndrome.</td><td>Human Rat</td><td>0.98</td><td>1.12</td><td><strong>2.37</strong></td><td>[120]</td></tr><tr><td></td><td>RCVRN (RCV1)</td><td>Recoverin</td><td>Calcium ion binding activity</td><td>TM4SF4 is an emerging molecular target for anticancer therapy against cancer phenotypes and chemoresistance.</td><td>Human</td><td>0.75</td><td><strong>2.13</strong></td><td><strong>2.26</strong></td><td>[121]</td></tr><tr><td></td><td>TM4SF4</td><td>Transmembrane 4 L6 family member 4</td><td>Signal transduction</td><td>Abnormally expressed in glioma compared to normal brain tissues. Involved in the development and progression of malignant tumors.</td><td>Human</td><td><strong>1.78</strong></td><td><strong>1.74</strong></td><td><strong>2.13</strong></td><td>[122]</td></tr><tr><td></td><td>ZDHHC23</td><td>zDHHC palmitoyltransferase 23</td><td>Protein-cysteine S-palmitoyltransferase</td><td>Involved in the development and progression of malignant tumors. Abcb9 is expressed by Sertoli cells.</td><td>Human Yellow</td><td>0.67</td><td><strong>2.98</strong></td><td>1.67</td><td>[123,124]</td></tr><tr><td></td><td>ABCB9</td><td>ATP binding cassette subfamily B member 9</td><td>ABC transporter</td><td></td><td>Human Mouse</td><td>0.26</td><td>-1.34</td><td>-0.09</td><td>[125]</td></tr><tr><td></td><td>BAZ1B</td><td>Bromodomain adjacent to zinc finger domain 1B</td><td>Involved in chromatin-dependent regulation of transcription</td><td>May be a new target for endocrine therapy-resistant breast cancer.</td><td>Human</td><td>0.28</td><td>-0.37</td><td>-2.62</td><td>[126]</td></tr><tr><td></td><td>FBXL16</td><td>F-box and leucine rich repeat protein 16</td><td>Part of SCF ubiquitin ligase complex</td><td>Controls tamoxifen sensitivity via regulation of mitochondrial respiration in estrogen receptor-positive breast cancer cells. Inhibits lung cancer metastasis by regulating the Wnt/β-catenin signaling pathway.</td><td>Human</td><td>-0.17</td><td>-1.64</td><td>-1.01</td><td>[127]</td></tr><tr><td></td><td>TMEM196</td><td>Transmembrane protein 196</td><td>Transmembrane protein</td><td>Controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC.</td><td>Mice</td><td>-2.42</td><td>0.8</td><td>-1.23</td><td>[128]</td></tr><tr><td></td><td>AAMDC</td><td>Adipogenesis associated Mth938 domain containing</td><td>Transcription factor</td><td>Regulates transcription by docking onto phosphorylated serine-2 in the C-terminal domain of Pol II.</td><td>Human Mice</td><td>0.19</td><td>1.5</td><td>1.1</td><td>[129]</td></tr><tr><td></td><td>PHF3</td><td>PHD finger protein 3</td><td>Transcription factor</td><td>Related to neurodevelopmental abnormalities, intracellular vesicle trafficking and signaling pathways related to cancer cell behavior.</td><td>Human</td><td>1.09</td><td>1.4</td><td>0.71</td><td>[130]</td></tr><tr><td rowspan="4">Other</td><td>RAB39B</td><td>RAB39B, member RAS oncogene family</td><td>Small GTPase involved in vesicular trafficking.</td><td>The Ric1-Rgp1 complex is a guanine nucleotide exchange factor. Involved in the stability and assembly of the outer mitochondrial membrane complex.</td><td>Human</td><td>0.99</td><td>1.41</td><td>0.96</td><td>[131]</td></tr><tr><td>RIC1</td><td>RIC1 homolog, RAB6A GEF complex partner 1</td><td>Guanyl-nucleotide exchange factor and small GTPase binding</td><td>Disruption of the mouse Jhy causes</td><td>Human</td><td><strong>2.26</strong></td><td>1.41</td><td>1.55</td><td>[132]</td></tr><tr><td>TOMM6 (TOM6)</td><td>Translocase of outer mitochondrial membrane 6</td><td>Protein transport</td><td></td><td>Human</td><td>1.17</td><td>1.07</td><td><strong>2.44</strong></td><td>[73]</td></tr><tr><td>XP_038635116</td><td>jhy protein homolog</td><td>Junctional cadherin complex regulator</td><td></td><td>Mice</td><td>0.87</td><td>0.51</td><td><strong>4.66</strong></td><td>[70]</td></tr></tbody></table>

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<table><caption>**Table 2 (continued)**</caption><thead><tr><th rowspan="2">Keyword</th><th rowspan="2">DAPs</th><th rowspan="2">Full name</th><th rowspan="2">Function</th><th rowspan="2">Potential significance in spermatogenesis (if any)</th><th rowspan="2">Species</th><th colspan="3">Log2(ratio)</th><th rowspan="2">Reference</th></tr><tr><th>ScGPA2</th><th>ScGPB5</th><th>ScGPB5-ScGPA2</th></tr></thead><tbody><tr><td>XP_038639553</td><td></td><td>Non-histone chromosomal protein HMG-14-like</td><td>Chromatin binding activity</td><td>abnormal ciliary microtubule patterning and juvenile hydrocephalus. Involved in nucleotide-excision repair, NAD+ ADP-ribosyltransferase activity, and response to UV. Located in female germ cell nucleus. Predicted to enable ATPase binding activity, calcium ion binding activity, and transmembrane transporter binding activity.</td><td>Human Mice</td><td>2.16</td><td>1.18</td><td>3.03</td><td>NCBI Gene</td></tr><tr><td>XP_038639919</td><td></td><td>Sarcoplasmic reticulum histidine-rich calcium-binding</td><td>Calcium binding</td><td>Predicted to be involved in Golgi to plasma membrane protein transport and positive regulation of axon extension. Predicted to be involved in several processes, including cilium assembly and regulation of cilium beat frequency. Vitamin K-dependent plasma protein functions as a cofactor for the anticoagulant protease APC.</td><td>Human Mice</td><td>-0.87</td><td>1.84</td><td>-0.3</td><td>NCBI Gene</td></tr><tr><td>XP_038648476</td><td></td><td>Golgin subfamily A member 4-like</td><td>GTPase binding activity</td><td>ZFP462 KO animals showed nuclear chromatin disorders. AGO1 represses H3K9me2 mark that appears at R-loop-forming terminator regions.</td><td>Human Mice</td><td>0.83</td><td>1.08</td><td>2.03</td><td>NCBI Gene</td></tr><tr><td>XP_038663118</td><td></td><td>Dynein assembly factor 1, axonemal isoform X18</td><td>Dynein complex binding activity</td><td>Participates in the proton/pH gradient required for membrane trafficking.</td><td>Human Mice</td><td>0.39</td><td>3.05</td><td>1.05</td><td>NCBI Gene</td></tr><tr><td>XP_038675849</td><td></td><td>Vitamin K-dependent protein S</td><td>Cofactor for the anticoagulant protease</td><td>CTNNBIP1 is a suppressor of the Wnt/β-catenin signaling.</td><td>Human Mice</td><td>-0.06</td><td>2.3</td><td>0.45</td><td>NCBI Gene</td></tr><tr><td>ZNF462</td><td></td><td>Zinc finger protein 462</td><td>Transcriptional regulation</td><td>ERAL1 serves as a mitochondrial rRNA chaperone which facilitates lysine 63 (K63)-linked ubiquitination of RIG-I/MDA5. Is involved in the cleavage of the monosaccharides N-acetylglucosamine and N-acetylgalactosamine. A transporter of Mg<sup>2+</sup> and, to a lesser extent, Fe<sup>2+</sup>, Zn<sup>2+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup> and Cd<sup>2+</sup> across the plasma membrane of epithelial cells.</td><td>Xenopus</td><td>1.8</td><td>2.72</td><td>4.48</td><td>[72]</td></tr><tr><td>AGO1 (EIF2C1)</td><td></td><td>Argonaute RISC component 1</td><td>RNA interference and RNA silencing</td><td>ERAL1 serves as a mitochondrial rRNA chaperone which facilitates lysine 63 (K63)-linked ubiquitination of RIG-I/MDA5. Is involved in the cleavage of the monosaccharides N-acetylglucosamine and N-acetylgalactosamine. A transporter of Mg<sup>2+</sup> and, to a lesser extent, Fe<sup>2+</sup>, Zn<sup>2+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup> and Cd<sup>2+</sup> across the plasma membrane of epithelial cells.</td><td>Human Mice</td><td>-0.32</td><td>-0.5</td><td>-1.85</td><td>[133]</td></tr><tr><td>ATP6V0C</td><td></td><td>ATPase H+ transporting V0 subunit c</td><td>Component of V-ATPase that mediates acidification organelles. Prevents interaction between CTNNB1 and TCF family members</td><td>CTNNBIP1 is a suppressor of the Wnt/β-catenin signaling.</td><td>Drosophila Human</td><td>-0.62</td><td>-1.32</td><td>-0.79</td><td>[134]</td></tr><tr><td>CTNNBIP1 (ICAT)</td><td></td><td>Catenin beta interacting protein 1</td><td>Assembly of the 28S ribosomal subunit</td><td>ERAL1 serves as a mitochondrial rRNA chaperone which facilitates lysine 63 (K63)-linked ubiquitination of RIG-I/MDA5. Is involved in the cleavage of the monosaccharides N-acetylglucosamine and N-acetylgalactosamine. A transporter of Mg<sup>2+</sup> and, to a lesser extent, Fe<sup>2+</sup>, Zn<sup>2+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup> and Cd<sup>2+</sup> across the plasma membrane of epithelial cells.</td><td>Mice</td><td>-0.38</td><td>-0.11</td><td>-2.04</td><td>[134]</td></tr><tr><td>ERAL1</td><td></td><td>Era-Like 12S mitochondrial rRNA chaperone 1</td><td>Assembly of the 28S ribosomal subunit</td><td>ERAL1 serves as a mitochondrial rRNA chaperone which facilitates lysine 63 (K63)-linked ubiquitination of RIG-I/MDA5. Is involved in the cleavage of the monosaccharides N-acetylglucosamine and N-acetylgalactosamine. A transporter of Mg<sup>2+</sup> and, to a lesser extent, Fe<sup>2+</sup>, Zn<sup>2+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup> and Cd<sup>2+</sup> across the plasma membrane of epithelial cells.</td><td>Human Mice</td><td>0.32</td><td>-2.08</td><td>-0.63</td><td>[135]</td></tr><tr><td>HEXD (HEXDC)</td><td></td><td>Hexosaminidase D</td><td>Carbohydrate metabolic process</td><td>ERAL1 serves as a mitochondrial rRNA chaperone which facilitates lysine 63 (K63)-linked ubiquitination of RIG-I/MDA5. Is involved in the cleavage of the monosaccharides N-acetylglucosamine and N-acetylgalactosamine. A transporter of Mg<sup>2+</sup> and, to a lesser extent, Fe<sup>2+</sup>, Zn<sup>2+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup> and Cd<sup>2+</sup> across the plasma membrane of epithelial cells.</td><td>Human Mice</td><td>0.31</td><td>-1.62</td><td>0.04</td><td>[136]</td></tr><tr><td>SLC41A1</td><td></td><td>Solute carrier family 41 member 1</td><td>Inorganic cation transmembrane transporter</td><td>ERAL1 serves as a mitochondrial rRNA chaperone which facilitates lysine 63 (K63)-linked ubiquitination of RIG-I/MDA5. Is involved in the cleavage of the monosaccharides N-acetylglucosamine and N-acetylgalactosamine. A transporter of Mg<sup>2+</sup> and, to a lesser extent, Fe<sup>2+</sup>, Zn<sup>2+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup> and Cd<sup>2+</sup> across the plasma membrane of epithelial cells.</td><td>Mice</td><td>-0.47</td><td>-0.38</td><td>-1.81</td><td>[137]</td></tr></tbody></table>

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<table><caption>**Table 2 (continued)**</caption><thead><tr><th rowspan="2">Keyword</th><th rowspan="2">DAPs</th><th rowspan="2">Full name</th><th rowspan="2">Function</th><th rowspan="2">Potential significance in spermatogenesis (if any)</th><th rowspan="2">Species</th><th colspan="3">Log2(ratio)</th><th rowspan="2">Reference</th></tr><tr><th>ScGPA2</th><th>ScGPB5</th><th>ScGPB5-ScGPA2</th></tr></thead><tbody><tr><td></td><td>SULT1C2</td><td>Sulfotransferase family 1C member 2</td><td>Sulfate conjugation of many compounds</td><td>Sulfate conjugation of various substrates and xenobiotics in the detoxification pathway.<br/>Is a GEF for the RHOA and RAC1 GTPases involved in actin remodeling necessary for cell migration and growth.</td><td>Human</td><td>-0.02</td><td>-1.45</td><td>-0.61</td><td>[138]</td></tr><tr><td></td><td>TRIO</td><td>Trio Rho guanine nucleotide exchange factor</td><td>GDP to GTP exchange factor</td><td>Is a GEF for the RHOA and RAC1 GTPases involved in actin remodeling necessary for cell migration and growth.<br/>Involved in cell morphology and mitotic progression.<br/>Involved in enteric smooth muscle relaxation, water/electrolytes/gastric acid, insulin and glucagon release.</td><td>Fly, Mice<br/>Rat, Worm</td><td>-0.68</td><td>-0.76</td><td>-3.21</td><td>[71]</td></tr><tr><td></td><td>XP_038633100</td><td>A-kinase anchor protein 2-like</td><td>Actin-bundling protein</td><td>Involved in enteric smooth muscle relaxation, water/electrolytes/gastric acid, insulin and glucagon release.<br/>The encoded protein forms a triple helical heterotrimer that assembles into a type IV collagen network.<br/>Present in genitourinary system, hemolymphoid system, nervous system, respiratory system, and sensory organ.</td><td>Human<br/>Mice</td><td>0.08</td><td>-1.5</td><td>0.02</td><td>NCBI<br/>Gene</td></tr><tr><td></td><td>XP_038639276</td><td>Vasoactive intestinal polypeptide (VIP) peptides-like</td><td>Neuropeptide of the glucagon/secretin superfamily</td><td>Involved in enteric smooth muscle relaxation, water/electrolytes/gastric acid, insulin and glucagon release.<br/>The encoded protein forms a triple helical heterotrimer that assembles into a type IV collagen network.<br/>Present in genitourinary system, hemolymphoid system, nervous system, respiratory system, and sensory organ.</td><td>Human<br/>Mice</td><td>0.16</td><td>-1.5</td><td>-0.5</td><td>NCBI<br/>Gene</td></tr><tr><td></td><td>XP_038648791</td><td>Collagen alpha-1(IV) chain-like</td><td>Alpha-1 subunit of the type IV collagens</td><td>Involved in enteric smooth muscle relaxation, water/electrolytes/gastric acid, insulin and glucagon release.<br/>The encoded protein forms a triple helical heterotrimer that assembles into a type IV collagen network.<br/>Present in genitourinary system, hemolymphoid system, nervous system, respiratory system, and sensory organ.</td><td>Human<br/>Mice</td><td>-0.4</td><td>-1.64</td><td>-0.5</td><td>NCBI<br/>Gene</td></tr><tr><td></td><td>XP_038650721</td><td>Microtubule organization protein AKNA-like</td><td>DNA binding</td><td>Involved in enteric smooth muscle relaxation, water/electrolytes/gastric acid, insulin and glucagon release.<br/>The encoded protein forms a triple helical heterotrimer that assembles into a type IV collagen network.<br/>Present in genitourinary system, hemolymphoid system, nervous system, respiratory system, and sensory organ.</td><td>Human<br/>Mice</td><td>-1</td><td>-1.32</td><td>-0.6</td><td>NCBI<br/>Gene</td></tr><tr><td rowspan="5">Uncharacterized</td><td>XP_038638443</td><td>Uncharacterized protein</td><td>NA</td><td>NA</td><td>NA</td><td>1.22</td><td>1.52</td><td>2.19</td><td>-</td></tr><tr><td>XP_038663965</td><td>Uncharacterized oxidoreductase YjmC-like</td><td>NA</td><td>NA</td><td>NA</td><td>-0.29</td><td>1.93</td><td>0.17</td><td>-</td></tr><tr><td>XP_038651296</td><td>Uncharacterized protein</td><td>NA</td><td>NA</td><td>NA</td><td>-0.08</td><td>-1.18</td><td>-0.65</td><td>-</td></tr><tr><td>XP_038654653</td><td>Uncharacterized protein</td><td>NA</td><td>NA</td><td>NA</td><td>-0.44</td><td>-3.46</td><td>-1.44</td><td>-</td></tr><tr><td>XP_038661993</td><td>Uncharacterized protein</td><td>NA</td><td>NA</td><td>NA</td><td>-0.12</td><td>-1.29</td><td>-0.62</td><td>-</td></tr><tr><td></td><td>XP_038673256</td><td>Guanylate-binding protein 1-like</td><td>NA</td><td>NA</td><td>NA</td><td>-0.48</td><td>-1.73</td><td>-1.23</td><td>-</td></tr></tbody></table>

ranging from 3.33 to 2.44 (Table 2). Among the 32 DAPs whose abundance was strongly decreased under the ScGPB5 treatment, the 10 most significant variations, with a Log2ratio ranging from -4 to -1.73 according to the DAPs, were observed for TMEM164, XP_038654653, IRGC, H2-AA, ERAL1, FILIP1, TRP53INP1, CCDC38, MRPS16, and XP_038673256 (Table 2). Finally, of the 29 DAPs that were highly increased in abundance by the ScGPB5#ScGPA2 treatment, the 10 most significant variations, with a Log2ratio ranging from 4.66 to 3.40 according to the DAPs, were observed for XP_038635116, ZNF462, SPINK2, SPCS3, COX7B, NPHS1, LRRC69, GEM, XPC, SLC7A1, and CAPN3 (Table 2). Among the 15 DAPs whose abundance decreased significantly under the ScGPB5#ScGPA2 treatment, 10 of them showed the most significant variation, with a Log2ratio ranging from -4.45 to -2.08. These DAPs were TMEM164, SLC25A28, FUT2, TRIO, ZMYM4, BAZ1B, NSUN6, OSBPL6, RASEF, PRKG1 (Table 2).

## 4. Discussion

Our previous study showed that GPA2, GPB5, and GPA2/GPB5 are potential paracrine regulators of testicular function in catsharks. The corresponding recombinant proteins activated FSHR, LHR, and TSHR, all of which are present in the zone of late spermatids [4,7]. Thus, the present study aimed to explore their biological functions through a

proteomic analysis of testicular explants treated with recombinant ScGPA2, ScGPB5, or single-chain ScGPB5#ScGPA2.

According to the results of the ANOVA, the recombinant proteins may have distinct biological functions. Based on the number of DAPs identified, ScGPB5 and ScGPB5#ScGPA2 appear to be more efficient than ScGPA2. However, due to experimental constraints, we were unable to stimulate the explants with the EC50 concentrations, which may have contributed to the observed differences between treatments. Despite this question on the relative role of each recombinant protein, 103 highly modulated DAPs have been evidenced, with a Log2ratio ranging from 1.09 to 4.66 for the increased DAPs and from -1.18 to -5.48 for the decreased DAPs. This corresponds to a difference in abundance ranging from 2 to 44-fold. To gain biological insights regarding identified proteins, functional annotation was performed using the NCBI database and systematic literature searches. Obtained information allowed us to distribute DAPs according to representative keywords. Of the 103 DAPs, 56 were associated with reproduction (subanalysis groups: spermatids; sperm motility; infertility/subfertility; spermatogenesis or oogenesis; and testicular function); 17 were associated with immunity/disease subanalysis groups; and 30 were associated with other/uncharacterized subanalysis groups. This distribution of DAPs according to these different analysis groups aligns with literature on the functions of GPA2 and GPB5 in regulating gametogenesis
[14,16,17,31], and immune response [13,32]. The results of this analysis also highlight new candidate proteins associated with spermatogenesis.

All three treatments resulted in significant modulation of DAPs related to reproduction. Among the DAPs whose abundance increased were proteins associated with mitochondria such as the ATP synthase ATP5MK, which could be involved in sperm mitochondrial dehydrogenase (diaphorase) activity [33]; COX7B which is part of the respiratory chain complex IV and whose importance in sperm motility has been demonstrated in Cox7b2 KO mice [34]; as well as the interferon inducible protein IFITM3 which has been localized on the head and mitochondria of spermatozoa and that may increase sperm motility in the mouse [35]. Other DAPs were the glucosidase GANC which is able to release glucose from long substrates such as the glycogen [36] and the D-glucose transmembrane transporter SLC2A1 (GLUT1). Glucose is beneficial for optimal capacitation of human sperm by enhancing ATP production [37]. Dysregulation of GLUT1, or of other testicular GLUTs, can potentially lead to male infertility [38]. DAPs related to acrosome were the signal peptidase complex SPCS3 related to the acrosomal and plasma membranes which function was stimulated by vitamin E in the sheep [39], and the serine peptidase inhibitor SPINK2. SPINK2 is of particular interest due to its acrosin inhibitor function involved in acrosome protection. The absence of SPINK2 induced by protease stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to arrest of spermatid differentiation in mice [40]. The kinesin KIF16B (kinesin-3 family) was also increased in abundance but despite its detection in mice testes [41], its involvement in spermiogenesis has not been reported [42] while another member of the kinesin-3 family, KIFC1, has been associated to globozoospermia in man [43] and to acrosome formation and nuclear shaping during spermiogenesis in the lobster [44]. DAPs involved in DNA repair such as the topoisomerase binding protein BTBD2, XP_038639553 and XPC (p125/Sec23ip) which are involved in nucleotide-excision repair [45,46] were also increased in abundance. Sperm from p125-deficient mice had round heads and lacked the acrosome [45]. Other DAPs related to subfertility or infertility were: the molecular chaperone CRYAB which function in sperm viability remains to be explored since CRYAB was overrepresented in idiopathic infertile patients [47] but associated with sperm viability in the rooster [48]; EEF1A1 whose mutation resulted in infertility due to abnormal spermiogenesis [49]; LRRC69 which is significantly overrepresented in patients with spermatogenic failure [50]; and NPHS1 (Nephrin) whose low and irregular amount is observed in patients with hypospermatogenesis and Sertoli cell-only syndrome [51].

Conversely, several DAPs showed decreased abundance under treatments. Among these, the decreased abundance of the tetraspanin MS4A15 with ScGPB5 may be of particular interest, as the oligomerization of the MS4A family member, MS4A13-2, in sperm cell membranes affects their ability to fertilize [52]. Moreover, studies on mammalian cell lines have shown that MS4A15, which is located in the endoplasmic reticulum, blocks ferroptosis by depleting luminal Ca²⁺ stores and converting membrane phospholipids into ferroptosis-resistant form [53]. In our results, the three treatments have significantly decreased the abundance of TMEM164 which promote ferroptotic cell death by selectively mediate ATG5-dependent autophagosome degradation of ferritin, glutathione peroxidase 4 and lipid droplets [54]. The roles of MS4A15 and TMEM164, which are opposite in controlling ferroptosis, remains to be further explored during spermiogenesis. The abundance of the mitochondrial iron transporter SLC25A28 (Mitoferrin-2) was also significantly decreased by ScGPB5#ScGPA2 and loss of mitoferrin-2 results in decreased male fertility [55]. Other DAPs that could be related to spermiogenesis are the reader CCDC38, predicted to be involved in acrosome assembly and sperm flagellum assembly [56], FILIP1 that promotes the degradation of the filamin A, which may be involved in the sperm tail axoneme formation [57], the immunity-related GTPases IRGC1, involved in the correct formation of the sperm fibrous sheath in mice [58], and the chloride transmembrane

transporter CLCN7 (CLC7) that may participate in sperm capacitation [59]. DAPs associated with gametogenesis include RYBP, a member of the polycomb repressive complex 1 involved in the monoubiquitination of histone H2A, which regulates meiosis and sperm motility in mice [60]. The abundance of AKAP17A, a protein involved in the regulation of pre-mRNA splicing, was also decreased with ScGPB5#ScGPA2. AKAP17A has previously been detected in the testis [61] and is overrepresented in the testes of fetuses with fetal Klinefelter syndrome [62]. Of DAPs associated with subfertility/infertility, our results highlighted the significant decrease in abundance of the fucosyltransferase FUT2 [63], the intracellular lipid receptor OSBPL6 [64], the replication factor C complex component CHTF18 [65], and two potential markers of asthenozoospermia, ESF1 and ZMYM4 [66]. NSUN6 was categorized under this keyword based on Nsun7- mutations associated with sperm motility defects in men [67] and mice [68], as well as the emerging role of RNA regulators, including the NSUN writer family, in reproduction and development [69]. The role of NSUN6 in spermiogenesis remains to be characterized.

On the other hand, functional annotation highlighted processes related to the immune response, such as infiltration of immune cells (RIPOR3), defense response to viruses (XP_038643196), regulation of complement activation (XP_038652419), regulation of MHC class II (H2-AA) and immunoglobulin production (IGKV4-55). In addition, 11 other DAPs were associated to cellular disorder and were related to cancers, notably tumor-like germ cell growth (EXTL3), ovarian cancer (AGR3, COL6A3), breast cancer (RBM12B2, BAZ1B, FBXL16) and other cancer phenotypes (TM4SF4, ZDHHC23, ABCB9, TMTM196). Finally, 23 DAPs were unclassified due to the heterogeneity of associated functions. Among these, the five DAPs exhibiting the most important changes in abundance are discussed herein. Three were associated with cytoskeleton structures: jhy protein homolog (XP_038635116), showing a 25-fold increase, which induced abnormal microtubule organization in mice [70]; the triple functional domain protein (TRIO), whose abundance decreased by 9-fold, which is known to be involved in the remodeling of actin [71]; and the dynein axonemal assembly factor 1 (XP_038663118), showing a 8-fold increase. The remaining two DAPs were the zinc finger protein 462 (ZNF462), which increased 22-fold in abundance and is involved in chromatin integrity [72], and the mitochondrial import receptor subunit TOM6, which increased 5-fold in abundance and is involved in the stability and assembly of the outer mitochondrial membrane complex [73].

In conclusion, treatment with recombinant ScGPA2, ScGPB5, or single-chain ScGPB5#ScGPA2 induced differentially abundant proteins in testicular explants at stages of spermiogenesis of *S. canicula*. This work provides the first molecular insights into the roles of the glycoprotein hormones GPA2 and GPB5 during spermiogenesis. To further explore hormone pathway dynamics, the phosphoproteome may be studied using the method described by Huwa et al. [139]. As mentioned above, one limitation of our study was the inability to treat the explants at the EC50 concentration due to constraints in the production of recombinant hormones. This limits the interpretation of the differences observed between hormones. Similarly, we were unable to perform dose-response analyses, and we did not verify the glycosylation states of the recombinant hormones. Nevertheless, the characterization of these DAPs in an elasmobranch suggests conservation of the critical functions of GPA2 and GPB5 during spermatogenesis, and highlights their paracrine functions in addition to the well-known endocrine regulation mediated by gonadotropin hormones.

### CRediT authorship contribution statement

**Fabian Jeanne:** Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Writing – review & editing, Writing – original draft. **Stanislas Pilet:** Resources, Methodology, Investigation, Formal analysis. **Benoît Bernay:** Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization,
Writing – review & editing. **Pascal Sourdaine:** Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization, Writing – review & editing.

## Fundings

University of Caen Normandy [F.J.'s PhD grant], the European Regional Development Fund [Operational Program ERDF/ESF 20142020] under the project "Manche 2021 – Plateformes d'exploitation de ressources marines", the "Région Normandie" under the project "Emergent CauSy". UHPLC-MS/MS equipment was acquired as part of the Equip'innov Caen 2022 project – PRISMM V2.0 sub-project, cofinanced by the European Union, the Normandy Region as part of the ERDF/ESF 2014–2020 operational program and by the French State, as part of the 2015–2020 State-Basse-Normandy Region Plan Contract.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors are grateful to Gaëtan Schires and Sébastien Henry from the Marine Biological Station of Roscoff (Centre de Ressources Biologiques – CRB Sorbonne Université-EMBRC, France) for providing the catfishs and the care given to the animals. The authors are grateful to the University of Caen Normandy, the "Région Normandie", the French state and the European Union for fundings.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jprot.2026.105627.

## Data availability

Complete list of proteins with blast results and clusters for proteomic analysis (Table S1) (XLSX). Mass spectrometry data can be found online (https://www.iprox.cn/) with the data set identifier IPX0014104001.

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