Proteomic dynamics of bull sperm during post-testicular maturation
Inês Leites, Patrícia Diniz, Margarida Fardilha, Joana Santiago, Graça Ferreira-Dias, Luís Lopes-da-Costa, Elisabete Silva
TL;DR
This study maps changes in bull sperm proteins during maturation, revealing shifts in function and metabolism as sperm move through the epididymis.
Contribution
The study provides a detailed comparative proteomic profile of bull sperm at different maturation stages, revealing novel insights into sperm function and histone dynamics.
Findings
Sperm proteomes show distinct biological processes at each maturation stage, including post-transcriptional regulation in testicular sperm and OXPHOS in cauda epididymal sperm.
Histone replacement by protamines is not completed in the testis but continues through epididymal transit, with histone modifying enzymes detected in caput sperm.
The ubiquitin–proteasome system is significantly enriched in conserved sperm proteomes across species, suggesting a role in proteomic remodeling.
Abstract
Mammalian sperm maturation during epididymal transit is driven by sequential interactions with the epididymal environment allowing the acquisition, loss and modification of sperm protein content. We report a comparative proteomic profile of testicular, caput and cauda epididymal spermatozoa using shotgun proteomics. Analysis rendered 2,305 proteins in testicular sperm (TestSperm), 2,554 in caput epididymal sperm (CaputSperm) and 2,038 in cauda epididymal sperm (CaudaSperm), including 702, 483 and 314 unique proteins, respectively. Gene Ontology (GO) enrichment analysis of total proteomes and unique proteins from each population allowed us to map biological processes (BP) to spermatozoa with sequential degrees of maturation. TestSperm was mostly enriched in processes related to post-transcriptional regulation, that may be reminiscent mechanisms to regulate spermatogenesis and sperm…
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Figure 7- —Centro de Investigação Interdisciplinar em Sanidade Animal
- —https://doi.org/10.13039/501100001871Fundação para a Ciência e a Tecnologia
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Taxonomy
TopicsSperm and Testicular Function · Reproductive Biology and Fertility · Reproductive biology and impacts on aquatic species
Background
Spermatogenesis is a highly regulated event that occurs in the seminiferous tubules, during which germ cells differentiate into spermatozoa (SPZ), classically divided in the stages of spermatocytogenesis, meiosis and spermiogenesis. The latter stage involves the differentiation of round spermatids into SPZ, which are released into the lumen of seminiferous tubules [1]. This process involves chromatin condensation and replacement of the approximately 1 to 15% of histones by protamines, which prevents de novo gene transcription and protein translation, leaving SPZ transcriptionally silent [2–4]. Mammalian testicular SPZ are functionally immature since they lack progressive motility and fertilizing ability, with these competences being acquired progressively as SPZ advance throughout the epididymis [5]. The epididymis is anatomically divided in three segments: caput (or head), corpus (or body) and cauda (or tail). Epithelial cells from these segments are highly specialized and maintain a differentiated epididymal luminal microenvironment, promoting SPZ functional maturation in the proximal segments and their storage in a quiescent state in the distal segment [5]. As transcription in mature sperm cells is residual [6], proteome remodeling associated with maturation is driven by exposure to factors present in the epididymal luminal milieu [7]. As SPZ advance throughout the epididymis, they undergo biochemical and molecular modifications, progressively losing and acquiring proteins [8]. As a result, fully mature SPZ display molecular signatures that differ from those of immature ones [9]. However, SPZ maturation is a complex process, not yet fully understood.
Mass spectrometry (MS)-based proteomic methodologies generates robust protein datasets, enabling absolute and relative protein quantification and the comparison of sperm protein profiles [10], thus allowing the in depth analysis of the protein dynamic remodeling during SPZ maturation. Fertility is a multifactorial trait and proteomics may also show the potential to identify key protein biomarkers of male fertility [11]. Until present, most proteomic studies explored ejaculated bull sperm [12–17] and only a few studies targeted epididymal sperm [17–19]. Sperm longevity-related proteins mainly pertain to the metabolic pathways, with proteins present only in the cauda epididymal samples being enriched in the glycolysis/gluconeogenesis, pentose phosphate, and glutathione pathways, compared to ejaculated SPZ [17]. A study directed to proteins Enolase 1 (ENO1), Cytochrome b-c1 complex subunit 1 (UQCRC1) and 2 (UQCRC2), and Non-selective voltage-gated ion channel VDAC2, concluded that the decline in SPZ motility and bull fertility may arise from an abnormal expression of mitochondrial proteins during epididymal maturation, which may induce reactive oxygen species (ROS) damage and premature energy consumption [19]. The aim of the present study was to evaluate bull SPZ proteome dynamic remodeling along the epididymal segments, associated with maturation and the acquisition of SPZ competence.
Methods
Sperm and epididymal tissue sample collection
Reproductive tracts from 5 bulls (ages between 18 and 24 months, crossbred) were recovered postmortem in a local slaughterhouse and transported at 4 °C to the laboratory within 2 h. Following cleaning with 70% ethanol, the testes and the epididymides were separated by dissection. Several sections across the albuginea exposed the testicular parenchyma, and testicular SPZ were recovered after sectioning the parenchyma, infusion of saline (NaCl 0,9%) and gentle squeezing. The epididymis was separated into its 3 segments, SPZ from the caput were recovered using saline infusion, and cauda SPZ recovered using a flushing technique [20]. Collected testicular and epididymal fluids were centrifuged for 1 min at 300 × g at room temperature (RT) to remove larger debris, the supernatant collected and centrifuged for 10 min at 300 × g, and the sperm pellet washed twice in PBS. To remove contaminant cells, sperm pellets were first incubated with Red Blood Cell Lysis buffer (155 mM NH_4_Cl, 12 mM NaHCO_3_, 0.1 mM EDTA, Sigma) for 2 min, immediately diluted with PBS and centrifuged at 400 × g for 10 min. The resulting pellet was washed with PBS, incubated with Somatic Cell Lysis Buffer (0.1% SDS, 0.5% Triton-X, Sigma) for 15 min on ice with gentle agitation, and centrifuged at 400 × g for 10 min. The resulting pellet was washed twice with PBS to remove any residual buffer. An aliquot was taken to evaluate purity (> 90%), concentration was determined and approximately 10 million sperm cells were sampled. An aliquot was also taken, centrifuged onto slides and spermatozoa were stained with nuclear staining (Hoechst, Sigma) to further confirm the purity level. These final samples were pelleted down and snap-frozen until proteomic analysis.
Sample preparation for proteomic analysis
Samples were resuspended lysis buffer (10% SDS, 100 mM triethylammonium bicarbonate (TEAB). Lysates were sonicated (PIXUL® Multi-Sample Sonicator, Active Motif) for 20 min (Pulse 50 cycles, PRF 1 kHz, Burst Rate 20 Hz). Protein concentration was measured by bicinchoninic acid (BCA) assay (Thermo Scientific) and 100 µg of protein were reduced and alkylated (10 mM Tris (2-carboxyethyl) phosphine hydrochloride, 40 mM chloroacetamide) and incubated (10 min, 95 °C in the dark). Phosphoric acid (2.75% final concentration) was added, and samples were diluted with binding buffer (90% methanol, 100 mM TEAB). Samples were loaded to S-Trap™ Micro Column (Protifi) and washed with binding buffer using centrifugation (1 min, 4,000 × g). Protein digestion was performed by adding TEAB (50 mM) containing trypsin (overnight at 37 °C). Peptides were eluted sequentially with 50 mM TEAB, then with 0.2% formic acid (FA) in water and finally with 0.2% FA in water/acetonitrile (ACN) (50/50, v/v), using centrifugation (1 min at 4,000 × g). Eluted peptides were dried by vacuum centrifugation and dissolved in TEAB (50 mM) then Peptide-N-Glycosidase F (PNGaseF) (40 IUBMB mU) was added for deglycosylation (37 °C, overnight). Peptides were acidified with trifluoroacetic acid (TFA) and desalted on reversed phase C18 OMIX tips (Agilent). Samples were loaded into the tips and, after binding, peptides were eluted twice with elution buffer (0.1% TFA in water/ACN (40:60, v/v)), transferred to HPLC inserts and dried in a vacuum concentrator.
Liquid Chromatography tandem mass spectrometry (LC–MS/MS) analysis
LC–MS/MS analysis was performed using an UltiMate™ 3000 ProFlow™ nanoLC system (Thermo Scientific) with in-line connected to a Orbitrap Exploris™ 240 Mass Spectrometer (Thermo Scientific) equipped with pneu-Nimbus dual ion source (Phoenix S&T). Peptides were dissolved in loading solvent (0.1% TFA in water/ACN (99.5:0.5, v/v), loaded into a PepMap™ Neo Trap cartridge (Thermo Scientific) and trapping was performed at 20 μl/min for 2 min. Peptides were separated on a Odyssey™ Ultimate™ (250 mm × 75 µm C18) (IonOpticks) kept at 45 °C, and eluted by applying a MS solvent (0.1% FA in ACN): 26.4% (80 min), 44% (95 min), 56% (100 min), followed by a wash with 56% MS solvent (5 min) and re-equilibration with 0.1% FA in water. The flow rate was set to 250 nl/min. The mass spectrometer was operated in data-independent mode, automatically switching between MS and MS/MS acquisition. MS spectra, ranging from 375–1500 m/z, were acquired with a normalized target value of 300%, a maximum fill time of 25 ms, resolution of 60,000 and followed by isolations with a precursor isolation width of 10 m/z for HCD fragmentation at an NCE of 30% after filling the trap at a normalized target value of 2000% for maximum injection time of 45 ms. MS/MS spectra were acquired at a resolution of 15,000 with a scan range of 200–1800 m/z in the Orbitrap analyzer. Isolation intervals were set from 400–900 m/z with a width of 10 m/z. The polydimethylcyclosiloxane background ion at 445.120028 Da was used for internal calibration and QCloud to control instrument longitudinal performance [21, 22].
Data analysis
Analysis of the mass spectrometry data was performed in Dia-NN (version 1.9) [23]. Precursor false discovery rate was set at 1%. Spectra were searched against the Bos taurus protein sequences in the Uniprot database (www.uniprot.org; database release version of 2024_01) [24], containing 23,836 sequences. Enzyme specificity was set as C-terminal to arginine and lysine and a maximum of 2 missed cleavages was tolerated. Variable modifications were set to oxidation (methionine) and acetylation (N-terminus) while fixed modifications were set to carbamidomethylation (cysteine). Searching parameters also included a 400–900 m/z precursor mass range filter and MS and MS/MS mass tolerance was set to 10.0 and 20 ppm, respectively.
DIA-NN output was filtered at a precursor and protein library q-value cut-off of 1% and only proteins identified by at least two proteotypic peptides were retained. iBAQ intensity columns were then added to the matrix using the DIAgui’s R package get_IBAQ function [25]. Outliers were identified by Interquartile Range method. PG MaxLFQ (Label Free Quantification) intensities were log2 transformed. Proteins with multiple accessions were grouped in a protein group. One representative accession/protein was chosen for the protein group. Proteins with less than 3 valid values in at least one group were removed and missing values were imputed from a normal distribution centered around the detection limit (package DEP [26]). To compare protein abundance between pairs of sample groups, statistical testing for differences between two group means was performed, using the package limma [27]. Only proteins present in both sample groups were considered differentially abundant. Statistical significance for differential regulation was set to a false discovery rate (FDR) of < 0.05 and |log2 FC|= 2.
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD066938.
Enrichment analysis
Quantified proteins and differentially abundant proteins (DAPs) were subjected to an Enrichment Analysis using a Functional Annotation Tool (DAVID) [28, 29]. The organism was set to Bos taurus and the Uniprot accessions were imputed in the query. Gene Ontology (GO) terms for Biological Processes (BP) were queried and the significance threshold considered was an adjust p value by the Benjamin-Hochberg method and inferior to 0.05. Furthermore, proteins from the different sperm populations were classified based on their protein class using PANTHER (Protein Analysis Through Evolutionary Relationships) Classification System (version 19.0) [30].
Graphic representations (Principal Component Analysis—PCA, volcano plots, GO bar and bubble plots) were performed using SRPlots [31]. Venn diagrams were performed using Venny 2.1 [32].
Results
Characterization of the testicular and epididymal bull sperm proteome
Following LC–MS/MS protein analysis of enriched sperm populations of the testis, and the caput and cauda segments of the epididymis, from the original 15 biological replicates, those that were considered outliers (n = 3) were excluded. Therefore, the final analysis included 3 biological replicates for testicular SPZ (TestSperm), 4 for caput epididymal SPZ (CaputSperm) and 5 for cauda epididymal SPZ (CaudaSperm). This rendered 9,593 identified proteins (FDR < 1% on the precursor and protein level) and 7,974 quantified proteins. For further analysis, only proteins with at least 2 unique peptides in each biological replicate were considered as well as proteins with valid LFQ values in at least 3 biological replicates of each group. These imposed conditions rendered 2,305 quantified proteins in TestSperm, 2,554 in CaputSperm and 2,038 in CaudaSperm (Supplementary Datafile S1). The average number of peptides per protein was 11.5, 12 and 11.6 in TestSperm, CaputSperm and CaudaSperm, respectively.
The PCA analysis revealed a clear clustering of the biological replicates assigned to each sperm population, explained by the PC2, which accounts for 28.9% of the observed variance. The PCA also revealed that the proteomes of the three SPZ populations are distinct from each other with PC1 accounting for 52.6% of the variance (Fig. 1A). A comparative analysis of the proteomic data, illustrated through a Venn diagram, revealed overlapping of proteins, with 1,106 being shared by the three SPZ populations. The analysis also revealed population-specific proteins: 702 in TestSperm, 483 in CaputSperm and 314 in CaudaSperm (Fig. 1B). Using Uniprot’s ID Mapping tool, the protein level of evidence was analyzed, revealing that the large majority had evidence at protein or transcript level (Fig. 1C).Fig. 1. Characterization of the proteome of bull testicular and epididymal spermatozoa. (A) PCA of the proteome of the three sperm populations (testicular, caput and cauda epididymal spermatozoa). (B) Venn diagram illustrates shared and unique proteins quantified in this study, across the three sperm populations. (C) Representation of the level of protein existence (deducted from UniProt annotations) across the three sperm populations
The analysis of each sperm population revealed that the majority of the identified proteins in the TestSperm population were metabolite interconversion enzymes and RNA metabolism proteins, whereas in the epididymal populations were protein modifying enzymes (Supplementary Figure S1).
The in silico analysis performed with GO annotations for “Cellular Component”, retrieved from Uniprot and EMBL-EBI (QuickGO) [33], showed that among all identified proteins, 1,541 proteins were mapped to specific domains in the sperm cell, being most of the proteins primarily mapped to the nucleus (775), mitochondria (414), and sperm tail (243) (Supplementary Figure S2).
Sperm proteome remodeling during epididymal maturation
The analysis of the 3 sperm populations showed the dynamic changes in SPZ protein content throughout the maturation process along the epididymal transit (Fig. 2). The comparison of the CaputSperm and TestSperm proteomes showed that 702 proteins were exclusively identified in TestSperm. On the other hand, the CaputSperm population contained 1,026 proteins that were not identified in the TestSperm population. Further along the epididymal transit, 905 proteins identified in CaputSperm were not identified in CaudaSperm. Conversely, 314 proteins were identified exclusively in CaudaSperm. The analysis also revealed that 1,106 proteins were maintained from the TestSperm population to the CaudaSperm.Fig. 2. Remodeling of sperm proteome during epididymal transit from caput to cauda. Graphical illustration of the epididymis representing acquired/gained proteins (green arrows) and removed/lost proteins (red arrows) from the spermatozoa in the caput and cauda epididymis
To go through the biological function of proteins identified in the 3 sperm populations, an enrichment analysis for BP was performed on the total proteome of each sperm population (Fig. 3 and Supplementary Datafile S2). Enrichment analysis of the total TestSperm proteome revealed that the most significantly enriched BP were related to RNA processing involved in the differentiation of germ cells as well as the terms ‘spermatogenesis’ and ‘spermatid development’. Enrichment analysis in both CaputSperm and CaudaSperm showed that the most significantly enriched BP were related to sperm motility and the binding of sperm to the zona pellucida.Fig. 3. Enriched biological process GO terms for SPZ total proteomes. Bubble plots representing the top 15 most significantly enriched biological process GO terms in total proteomes of the different sperm populations. (A) Testicular spermatozoa (TestSperm). (B) Caput epididymal spermatozoa (CaputSperm). (C) Cauda epididymal spermatozoa (CaudaSperm). Complete analysis is available in Supplementary Datafile S2
Enrichment analysis for BP was performed on the unique proteins identified in TestSperm (702), CaputSperm (483) and CaudaSperm (314). Bubble plots presented in Fig. 4 (and Supplementary Datafile S2), represent the most significant BP GO terms of each analysis. Specific TestSperm proteins revealed that the most significantly enriched terms were related to post-transcriptional regulatory mechanisms, such as RNA processing and splicing. Specific CaputSperm proteins most significantly enriched terms that included protein transport, complement activation and integrin signaling pathways. Specific CaudaSperm proteins significantly enriched term was mitochondrial respiratory chain complex I assembly.Fig. 4. Enriched biological process GO terms for specific proteins. Bubble plots representing the top significantly enriched biological process GO terms in specific (unique) proteins of each sperm population: TestSperm, CaputSperm and CaudaSperm. The complete analysis is available in Supplementary Datafile S2
Differentially abundant proteins involved in sperm post-testicular maturation
To identify differences in protein abundance, a comparative analysis was performed between the 3 sperm populations (Supplementary Datafile S3). The comparison between CaputSperm and TestSperm showed that 206 proteins were significantly upregulated in CaputSperm (Log_2_ FC ≥ 2; p ≤ 0.05), while 179 proteins were downregulated (Log_2_ FC ≤ −2; p ≤ 0.05) (Fig. 5A). Cylicin-2 (CYLC2; Log_2_ FC = 6.28), Acrosomal vesicle protein 1 (ACRV1; Log_2_ FC = 5.74) and Postacrosomal sheath WW domain-binding protein (PAWP; Log_2_ FC = 5.7) were the most upregulated proteins, whereas Pre-mRNA-splicing factor SYF1 (XAB2; Log_2_ FC = −6.29), Pericentriolar material 1 (PCM1; Log_2_ FC = −5.21) and Elongation factor 1-delta (EEF1D; Log_2_ FC = −5.08) were the most downregulated proteins in CaputSperm. To identify the BP related to the above changes in protein abundance, an enrichment analysis was performed, and results are shown in Fig. 5B. The most significant BP in upregulated proteins were related to sperm motility (‘flagellated sperm motility’, ‘cilium movement’), followed by sperm flagellum assembly and other microtubule-based processes, and also included ‘single fertilization’, as well as processes related to sperm energy metabolism. On the other hand, downregulated proteins were enriched in GO terms related to RNA processing and splicing.Fig. 5. Comparative proteomic analysis of DAPs across the three sperm populations. Volcano plots depicting DAPs between populations (adjusted p-value < 0.05) and bar graphs representing the 10 most significantly enriched biological process GO terms in the upregulated and downregulated proteins (red and blue bars, respectively). (A) and (B) CaputSperm vs. TestSperm. (C) and (D) CaudaSperm vs. TestSperm. (E) and (F) CaudaSperm vs CaputSperm. Complete list of DAPs and enrichment analysis is available in Supplementary Datafile S3
The comparative analysis between CaudaSperm and TestSperm revealed 198 significantly upregulated proteins in CaudaSperm (Log_2_ FC ≥ 2; p < 0.05), whereas many more proteins (419) were downregulated (Log_2_ FC ≤ −2; p > 0.05) (Fig. 5C). Cylicin-2 (CYLC2; Log_2_ FC = 6.32). Golgi associated RAB2 interactor protein-like/Rab2B-binding domain-containing protein (log_2_ FC = 6.16) and Cylicin-1 (CYLC1; log_2_ FC = 6.05) were the most upregulated proteins. Interleukin enhancer binding factor 2 (ILF2; log_2_ FC = −8.44), Interleukin enhancer binding factor 3 (ILF3; log_2_ FC = −8.06), and SURP and G-patch domain containing 2 (SUGP2; log_2_ FC = −7.83) were the most downregulated proteins. Enrichment analysis of up and downregulated proteins in CaudaSperm showed results close to those observed in CaputSperm, with BP of upregulated proteins of CaudaSperm being mainly related to sperm motility and energy metabolism (Fig. 5D).
Differential abundance analysis between CaudaSperm and CaputSperm proteomes identified one upregulated protein (Golgi associated RAB2 interactor protein-like; log_2_ FC = 2.57) and 310 downregulated proteins in the CaudaSperm. Fibrinogen alpha chain (FGA; log_2_ FC = −5.31), Heterochromatin protein 1-binding protein 3 (HP1BP3; log_2_ FC = −5.06) and Lamin B1 (LMNB1; log_2_ FC = −5.04) were the most downregulated proteins (Fig. 5E). Enrichment analysis of downregulated proteins revealed that they were mainly associated with RNA processing and splicing, as well as DNA repair (Fig. 5F).
Subproteomes involved in sperm maturation, fertilization and embryo development
In addition to enrichment analysis, an in silico analysis focused on specific GO terms, was used to identify proteins functionally related to sperm maturation, including motility, capacitation and acrosome reaction, fertilization and embryogenesis (Fig. 6 and Table 1).Fig. 6. Summary of proteins involved in key sperm functions/events. Graphical representation of the number of identified proteins in the current study involved in sperm motility, capacitation and acrosome reaction, fertilization, and embryo development, in the three sperm populations, recovered by an in silico analysisTable 1Summary of proteins involved in key sperm functions/events via in silico analysis.Sperm functionMotilityCapacitation/acrosome reactionFertilization****Embryo developmentGO IdentifiersGO:0030317, GO:1901317, GO:1901318, GO:1902093, GO:0007288, GO:0120316, GO:0048870GO:0060474, GO:0007340, GO:0060046, GO:0060478, GO:2000344, GO:0001675, GO:0048240GO:0007338, GO:0007339, GO:0007341, GO:0007342, GO:0007343, GO:0009566, GO:0035036, GO:0035037, GO:0080154GO:0043009, GO:0045995, GO:0001701, GO:0040019, GO:0001824, GO:0001825, GO:0001829, GO:0001832, GO:0001834, GO:0001835, GO:0007566, GO:0035039, GO:1901164, GO:1901165TestSperm, CaputSperm and CaudaSpermCABS1PACRGIQUBCFAP251CFAP77CIMIP2ATEKT4TSSK6NME8SPINLW1TEKT5TEKT2CFAP107TEKTL1DNALI1CCDC40RSPH6ALRRC23ROPN1LACTBL2LDHCCFAP210RIBC2IQCGARMC3FSIP2TEKT1LZTFL1CTNNA1SPAG8DNAH1CFAP52DNAJA1GARIN2AKAP3CFAP45SORDCFAP161CFAP276CFAP58EFHC2CFAP144CFAP53SPAG6EFHBQRICH2TPPP2TEX101ENKURROPN1CFAP95EFHC1NME7CFAP70PRSS55CFAP20ACTL7ACCDC136DLDGARIN3ZPBPROPN1LCABYRPRSS37TRIM36ACRBPSPACA1ROPN1ACTL9SPESP1IQCF1ACRCYLC1PRKACAACTL7ASPACA3CCT5ADAM2SPA17IZUMO1GARIN3CCT3ZPBPPRSS37ACRBPCLGNGLIPR1L1SPESP1PAWPTCP1HSPA1LCCT4ADAM32CCT7CCT2SPAG8CCDC136KLHL10VDAC2SPAM1TEX101ACTL9ADAM3ADisintegrin and metalloproteinase domain-containing protein 5-likePARK7CFAP70PRSS55CNOT1TRIP12TRIM28VIMNXNAKAP3MYH9RTCBEIF2S2LAMA1CFL1SOD1COL3A1COPS3PAWPPSMC4SLC25A20CaputSperm and CaudaSpermTEKT3RNASE10CCDC38CFAP97D1BBOF1DRC7CFAP141CCDC146CFAP206CFAP69CFAP43CCDC39CFAP61CFAP44CFAP68GAS8SPMIP10CFAP54SPMIP9SPEM1PIERCE1SPEF2SPMIP11CIMIP2CCFAP157PIERCE2TEKT3CFAP126TSSK4CFAP90CCDC42CCDC38ELSPBP1TMPRSS12ZPBP2GARIN1ALYZL4RNASE10ELSPBP1BBOF1CCDC146TMPRSS12AAASZPBP2SPACA4FAM170BMFGE8CD46LYZL6PLCZ1CD9TSSK4LAMA4RCN1TestSpermTMF1CEP192RFX2NECTIN2TNP2TMF1TBPL1TEX11TNP2KDM5BYBX3NECTIN2SYCP2DHX36WDR74CDK11SF3B6APLFBRCA1NMT1RBM46EMG1HORMAD1BYSLCTR9RRP7YBX3CaputSpermCFAP221RAC1AGTSPADH1H3.3UBE3ATRIP6LAMA3LAMA5CTCFMGAT1C1QBPSMARCB1H3.3CaudaSpermATP2B4CLXNPTENDEFB1EQTNIZUMO3Fibronectin type-II domain-containing proteinFAM170AOVCH2EQTNCRISP1TENT5CKEAP1
The analysis of GO annotations in testicular and epididymal sperm proteomes showed that 93 proteins were functionally related to the acquisition and maintenance of sperm motility (Fig. 6 and Table 1). Most of these proteins (n = 56) were present in testicular SPZ and maintained along maturation, whereas another set of proteins (n = 30) were identified in CaputSperm and CaudaSperm. Other proteins were specific to the TestSperm (TATA element modulatory factor 1 and Centrosomal protein 192), CaputSperm (Cilia and flagella associated protein 221 and Ras-related C3 botulinum toxin substrate 1) and CaudaSperm (Calaxin and Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN).
Regarding proteins functionally related to capacitation and acrosome reaction (n = 36), 18 were present in TestSperm and epididymal sperm. Only 6 proteins were specific to epididymal SPZ. TestSperm unique proteins included RFX2 (Regulatory factor X 2), NECTIN2 (Nectin cell adhesion molecule 2), TNP2 (Nuclear transition protein 2), TMF1 (TATA element modulatory factor 1) and TBPL1 (TATA box-binding protein-like 1), whereas the CaputSperm specific protein was AGT (Angiotensinogen), and CaudaSperm unique proteins included DEFB1 (Beta-defensin 1), EQTN (Equatorin), IZUMO3 (Izumo sperm-egg fusion protein 3) and Fibronectin type-II domain-containing protein.
A total of 64 proteins were functionally related to fertilization, including 33 proteins common to testicular and epididymal SPZ, and 16 acquired in the epididymis. A total of 50 proteins were functionally related to embryo development, from which 17 were identified in both testicular and epididymal SPZ 2 proteins were only identified in epididymal SPZ [LAMA4 and Reticulocalbin 1 (RCN1)]. A large set of proteins (n = 14) were exclusively identified in TestSperm, whereas 8 proteins were specific to the CaputSperm and only 2 to the CaudaSperm [TENT5C (Terminal nucleotidyltransferase 5 C) and KEAP1 (Kelch-like ECH-associated protein 1)].
Conserved species-specific epididymal sperm proteins
To evaluate the conserved status of the identified proteins, a comparative analysis with proteomes of epididymal SPZ was performed, using data published for mouse [34, 35], ram [36, 37] and pig [38, 39]. This analysis included 2,855 epididymal sperm proteins identified in the present study, 2,833 cauda epididymal sperm mouse proteins, 727 cauda epididymal sperm ram proteins, and 1,593 cauda epididymal sperm pig sperm proteins. Results showed that 264 proteins were shared between the 4 species (Fig. 7A and Supplementary Datafile S4). The GO enrichment analysis of BP of this set of conserved proteins showed that they were related to motility, sperm energy metabolism, proteasome related catabolic processes and fertilization (Fig. 7B). Proteins involved in motility include TEKT1/2/3, RSPH9, TSSK4, ROPN1L, IQUB, CCDC39, CCDC40, ROPN1, SORD (Sorbitol dehydrogenase) and Enkurin (ENKUR). Proteins involved in energy metabolism include PGAM1 and 2 (Phosphoglycerate mutase), ENO1/4 (Enolase 1 and 4), Fumarate hydratase (FH), ACAT1/2 (Acetyl-CoA acetyltransferase) and CPT2 (Carnitine O-palmitoyltransferase 2), hexokinase (HK1) and GPI (Glucose-6-phosphate isomerase). Proteins related to proteasome catabolic processes, including Proteasome subunit alpha type proteins (PSMA1/2/3/4/5/8), Transitional endoplasmic reticulum ATPase (VCP), calicin (CCIN) and UBX domain protein 11 (UBXN11). Proteins associated with fertilization included CCT2, CCT4, VDAC2, ZPBP, TEX101, ZPBP2, CCT5, SPA17, TCP1, ACTL9, SPESP1, TSSK4, ACRBP, ACTL7A and LYZL4.Fig. 7. Bull epididymal sperm proteins conserved among other mammalian species. (A) Venn diagram depicting shared epididymal sperm proteins (n = 264) from bull (present study, own proteomic data) and those published by others in ram, mouse and pig epididymal sperm. (B) Bar plot representing the top significantly enriched biological process GO terms in the conserved set of proteins. Complete list of conserved proteins and analysis is available in Supplementary Datafile S4
Histones and histone post-translational modifying proteins in post-testicular maturation
This study revealed that both testicular and epididymal sperm proteomes contain several histones and histone-modifying proteins. The present study also considered an in-depth analysis of the histone and histone-modifying proteins remodeling along epididymal maturation. The identified proteins included histones (H2A, H2B, H3 and H4), linker histones (H1) and variants (H1.1, H1.2, H1.3, H1.7, H1.8, H1.9, H1.10, macro.H2A.1, macro.H2A.2, H2AC18, H2AC10, H2B.K, H2A.L, H2B type 1-N, H2BC26, SubH2Bv, H3.3C, H3.3C-like, H3.4, CENP-A, H3.3) (Supplementary Datafile S5). Most of these histones were found in both testicular and epididymal SPZ, including the canonical core histones (H2A, H2B, H3, H4), their variants, and the Sperm protamine P2. Nevertheless, the histones H2B.K, H2A.L, H2B type 1-N, H2BC26, H3.2 and H3.3 were only found in CaputSperm. Several histones were not detected in CaudaSperm, including most of the H3 variants. As the proteomic analysis could not distinguish the post-translational modifications (PTMs) of histones, an in silico analysis was directed to the identification of enzymes involved in the histones PTMs, recovering proteins involved in acetylation of H4, phosphorylation of H2A, demethylation and deacetylation of H3 and H4 and methylation of H2A, H3 and H4 in epididymal sperm (Table 2). CaputSperm proteome included proteins for H2A modification (methylation and phosphorylation) but did not show proteins for H2B modification. For H3 and H4, we have identified modifying proteins involved in deacetylation, methylation and demethylation. Mature SPZ (CaudaSperm) appeared to have lost most of modifying proteins, being the most prevalent identified proteins involved in phosphorylation of H2A.Table 2. Summary of modifying enzymes related to histone PTMs in epididymal sperm via in silico analysis.TargetAcetylationDeacetylationMethylationDemethylationPhosphorylationCaputSpermH2A/H2A variantsPRMT5CARM1FBLPPEF1CDC14BPPP3CAPPP6CPPP1CBPPM1APPM1BDUSP21CTDNEP1PPP1CAPPP1CCDUSP18PTPN1PTPN6ACP1PGPH3/H3 variantsHDAC1PRMT5CARM1JMJD6KDM3BKDM1AH4/H4 variantsHDAC1PRMT5CARM1JMJD6CaudaSpermH2A/H2A variantsPRMT5FBLPPP2CBPPEF1CDC14BPPP6CPPP4CPPP1CBPPM1APPM1BDUSP21CPPED1CTDNEP1PPP1CAPPP3CCPPP1CCDUSP18ACP1PGPH3/H3 variantsSIRT3PRMT5H4/H4 variantsNAA50SIRT3PRMT5
Discussion
The present study considered a comprehensive proteome analysis of bull SPZ recovered from the testis and two epididymal segments, to characterize protein remodeling associated to SPZ maturation and competence, gained along the epididymal transit. Following spermatogenesis in the testis, SPZ are still immature, non-motile and not competent for fertilization. The first stages of SPZ maturation occur along epididymal transit and then during ejaculation when seminal plasma is added and is completed only in the female genital tract. During epididymal maturation, immature testicular SPZ progressively lose and acquire surface proteins, either permanently or transiently. These results indicate that SPZ undergo deep remodeling of their proteome, as 44% of the original (testicular and caput) proteins were not detected in CaudaSperm.
This study showed that TestSperm unique proteins are significantly enriched in BP GO terms including post-transcriptional regulatory mechanisms, such as mRNA splicing, mRNA capping, mRNA export and stability. The analysis identified 38 proteins involved in the spliceosome, which participate in the excision of noncoding introns from pre-mRNA to generate mature mRNA. Some of these proteins were previously described as playing a role in spermatogonia differentiation (YTHDC1, SNRPA1, CWF19L2) [40–42] and spermatid differentiation (RBM5, CWF19L2) [41, 43, 44]. The presence of RNA-binding proteins in testicular SPZ, which were not detected during the transition to the epididymis, indicates that they are remnants of spermatogenesis. Post-transcriptional regulatory mechanisms play important regulatory roles in mammalian spermatogenesis, particularly in cell transition to meiosis, as spliceosome dysregulation impairs sperm differentiation and maturation [40, 45–47]. Therefore, this study identified testicular sperm proteins that reflect regulatory mechanisms of spermatogenesis, not fully characterized, that could be explored as potential markers of male fertility.
In CaputSperm unique proteins, integrin-mediated signaling pathways stand among the most represented BP. Integrins, are a family of transmembrane cell adhesion molecules that participate in cell–cell and cell-extracellular matrix interactions [48]. Integrins have been implicated in several reproductive events, such as sperm migration through the female reproductive tract, sperm oviductal reservoir formation, oviductal fluid-sperm interaction, and gamete membrane adhesion and fusion [48]. The present study identified several sperm integrins and extracellular matrix (ECM) adhesive proteins that may interact with and regulate integrin activity (glycoprotein EMILIN1, laminin subunit alpha-5, laminin subunit alpha-3, galectin-3-binding protein, glycoprotein G, microfibril-associated glycoprotein 4 and FERMT2 protein). Although the SPZ-epididymosome interaction mechanism is not fully elucidated, it was proposed that bovine epididymosomes can fuse with SPZ to deliver cargo [49]. As integrins are also expressed in epididymosomes [50], authors hypothesize that SPZ-activated integrins, bound to SPZ-ECM adhesive proteins, participate in the fusion process, forming a complex that interacts with the integrins of the epididymosomes. A similar mechanism was proposed for the interaction between oviductosomes and SPZ membranes, which also involve tetraspanins [51]. Merc et al. (2021) suggested that this oviductosome-sperm interaction mechanism based on integrins and tetraspanins could be applied to SPZ-extracellular vesicle interactions in the male reproductive tract. Although the present study offers a valuable starting point, further validation studies are necessary to prove this hypothesis and to identify potential participants of the epididymosome-SPZ interaction. However, the possibility that SPZ integrins are involved in signaling to the epididymis cannot be excluded.
In CaputSperm we also identified unique proteins related to protein transport, which have been previously associated with endocytic pathways. This included components of the receptor-mediated endocytosis system, namely the Sorting Nexins (SNXs) and the Endosomal Sorting Complexes Required for Transport (ESCRT) [52], which are possibly involved in protein turnover, regulating the SPZ membrane remodeling. Although it is believed that SPZ lack the ability to perform endocytosis, the identification of these CaputSperm proteins suggests that SPZ may be equipped with a ‘minimal tool kit’, and that these endocytosis proteins may play other roles, such as signaling or vesicle fusion [53], namely in the uptake of epididymosomes and the release of their cargo [54].
The cauda epididymis segment stores functional mature SPZ in a metabolic quiescent state, protecting them against oxidative stress and complement-mediated attack [55]. Although bovine SPZ can use both glycolysis and OXPHOS to produce ATP, the latter is considered the primary metabolic pathway of ejaculated bull SPZ, supporting the energy demands of motility and fertility [56]. In the present study, CaudaSperm unique proteins were enriched in one single BP related to mitochondrial respiratory chain complex I (NDUFA8, NDUFB6, NDUFB11, NDUFB5, NDUFS5, NDUFC2, BCS1L, MT-ND2, MT-ND1). In fact, whereas glycolysis term is already enriched in TestSperm, mitochondrial activity related GO terms become significantly more enriched along the epididymis, with a higher significance in CaudaSperm. Pyruvate metabolism and tricarboxylic acid cycle, which feed the OXPHOS process, are also significantly enriched in this population. The results of the present study reflect the early findings that suggested that bovine epididymal SPZ energy metabolism is more glycolytic compared to ejaculate SPZ [17], in accordance with the concept that SPZ energy metabolism changes along the maturation process, becoming increasingly dependent on OXPHOS and less dependent on glycolysis, with mature SPZ being equipped for both. The total CaudaSperm proteome was also significantly enriched in terms related to oxidative stress response (GO:0006979) and glutathione metabolism (GO:0006749). These results further suggest that mature SPZ are not only capable of producing energy but are also equipped with machinery capable of mitigating the deleterious effects of ROS [57].
Concerning the proteins shared by the three sperm populations, DAP analysis showed that testis-specific proteins Cylicin-1, Cylicin-2 and ACRV1 were among the most upregulated proteins (log_2_FC > 5) in epididymal SPZ compared with testicular SPZ. Cylicin-1 and Cylicin-2 are components of the calyx, involved in acrosome assembly and stabilization [58, 59]. The ACRV1 is a component of the acrosomal matrix [60] and is implicated in the acrosome reaction and sperm-oocyte binding [61, 62]. The abundance of these proteins in epididymal SPZ, previously described as having testicular origin, likely reflects the protein remodeling during post-testicular maturation, in which the loss of other proteins makes them appear relatively more abundant in the proteome. However, we cannot exclude the possibility that these proteins are also acquired during post-testicular maturation.
This study performed an in silico comparative analysis of the total testicular and epididymal SPZ proteomes, focusing on proteins related to SPZ function. Most of the proteins involved in motility, acrosome reaction/capacitation, fertilization and embryo development were already detected in TestSperm and maintained during post-testicular maturation. Concerning motility, both testicular and epididymal SPZ populations, exhibit structural proteins related to flagellar assembly and stability, including axonemal and microtubule core components. Nevertheless, some flagellar structural proteins were exclusively found in epididymal SPZ. As maturation progresses along the epididymis, these structural proteins become tightly incorporated [63–65], thus more stabilized. Since in TestSperm, these proteins are loosely attached and not fully incorporated into the flagellum, the process of sperm purification and sample preparation might have led to loss of these proteins. Also, the exclusive presence of these proteins in epididymal SPZ, previously described as having testicular origin, might reflect the loss of other proteins (e.g. cytoplasmatic proteins) rendering some proteins relatively more abundant in this population [63].
Most of the shared testicular and epididymal proteins related to fertilization, are zona pellucida binding proteins (ZPBP, SPA17, TEX101, TCP1, CCT2, CCT3, CCT4, CCT5, CCT7, ADAM2, ADAM32, ADAM3A, and metalloprotease domain-containing protein 1A-like) or SPZ-oocyte fusion proteins (SPACA3, IZUMO1, SPAM1, SPESP1, and GLIPR1L1). The testicular origin of these proteins suggests that immature sperm contain most of the proteomic machinery required for fertilization, but the activity of these proteins maybe dependent on their modification and processing in the epididymis. For instance, some members of the ADAM family undergo proteolytic processing in the epididymis, involving the removal of pro- and metalloprotease domains, a process essential for their activity [66].
The shared testicular and epididymal proteins related to embryo development include those involved in epigenetic programming during the transition to blastomere and embryo implantation (TRIM28) [67, 68], as well as those involved in protection DNA integrity (COPS3, COP9 signalosome subunit 3), essential for early post-implantation survival [69]. These proteins are addressed as maternal and embryonic factors, but their role as paternal regulators of embryo development is not described. Therefore, the identification of these TestSperm embryo development related proteins aligns with the use of this SPZ population in Assisted Reproductive Technologies, like intra-cytoplasmic sperm injection, and suggests that they could serve as paternal biomarkers for the epigenetic quality and survival of early embryos.
To identify conserved mammalian SPZ proteins, this study compared the obtained bull epididymal SPZ proteomic data with published datasets from other species (ram, mouse and pig). This comparison rendered 264 common/conserved proteins. Interestingly, this analysis showed that the conserved proteins were significantly enriched in ubiquitin–proteasome catabolic processes. Ubiquitin–proteasome system (UPS) is a key pathway for protein degradation, and in epididymal SPZ, ubiquitination labels misfolded, damaged or excess proteins for degradation by the proteasome [70, 71]. The UPS was shown to play an important role in sperm motility [72], capacitation [73] and sperm-oocyte interactions [74]. In accordance with data from the mouse SPZ proteasome complex [75], the present study detected several components of the proteasome in testicular and epididymal SPZ populations. This includes components of the 26S proteasome: the 20S core (PSMA1-7 and PSMB1-7) and the 19S particle (PSMC1-6 and several other PSMD); components of the proteasome assembly chaperones (PSMG1 and 2), a proteasome inhibitor (PSMF1) and proteasome activators (PSME1-4). Overall, together with the presence of several ubiquitin ligases or E3 enzymes, results suggest that bull epididymal SPZ have the molecular machinery for protein ubiquitination to mark proteins for subsequent proteasomal degradation acting as a system for sperm quality control and eliminating defective sperm during maturation [76].
The proportion and localization of retained histones in mature SPZ influence fertility by modulating sperm function, fertilization and embryonic development [77]. For instances, perinuclear-derived somatic histones are retained in epididymal sperm possibly providing nuclear stabilization during the male pro-nucleus formation [78, 79]. Recent studies suggest that histone replacement by protamine is not fully completed in the testis and continues during the transit in the epididymis [80, 81]. Although the present study was not directed to histone identification and quantification, data evidenced changes in SPZ histone composition/abundance associated with post-testicular maturation. CaputSperm showed a higher diversity of histones compared to CaudaSperm, for instance H3 variants (H3.2, H3.3, H3.4) were only detected in CaputSperm. Studies in the mouse model suggest that H3.3 is involved in histone eviction and replacement with protamines [82], and that the amount of H3 decreases during transit from the caput to the cauda of the epididymis [80]. However, the absence of some histones/histone variants in CaudaSperm might reflect a lower abundance and/or undetected modified forms. In fact, as chromatin further compacts, histones become more difficult to access through protein extraction. Nevertheless, this different histone composition between Caput and Cauda warrant further investigation to elucidate their functional significance, and the identification of proteins associated with histone PTMs may provide information on SPZ epigenetic landscape. Histone PTMs appears more evident in CaputSperm, as also evidenced by the exclusive detection of H3 and H4 demethylases in Caput SPZ. Overall, results suggest that SPZ histone modifications are an integral part of the epididymal maturation process, showing different signatures between caput and cauda SPZ. This is in accordance with other reports [83–85], and associate SPZ histone PTMs to the increased chromatin condensation in mature SPZ, a mechanism that protects SPZ epigenetic information required for embryonic development, and to the balance between histone eviction and retention.
Conclusion
In conclusion, this study documented for the first time the dynamic remodeling of the bull SPZ proteome during the post testicular maturation along the epididymis. Results evidenced a deep turnover of SPZ protein content from the testis to the cauda epididymis, conferring specific signatures to the testis and epididymal segments. Histone abundance and diversity decrease along the epididymis, which may be related to SPZ chromatin compaction and epigenetic programming. The comparison with published epididymal SPZ proteomes of other species identified conserved proteins, mainly related to protein ubiquitination. In silico analysis identified testis and epididymal unique proteins involved in SPZ motility, capacitation, fertilization and embryo development. All these unique proteins show the potential to emerge as markers of SPZ competence and function and warrant further studies to evaluate their diagnostic value in fertility evaluation settings.
Limitations of the study
This proteomic data analysis defined the minimal criteria for protein identification as the presence of at least two unique peptides in each biological replicate, as well as valid LFQ values in at least three biological replicates per group. While these criteria reduce the noise and false positives, increasing the confidence of the analysis, they may also exclude low-abundance and condition-specific proteins (e.g. transiently expressed proteins). Specifically, the number of proteins acquired and lost by each SPZ population may be influenced by the above criteria, as it is not possible to rule out the possibility of missing low-abundance proteins. Furthermore, the process for sperm purification employed in the present study, using somatic cell and RBC lysis buffer might have contributed to loss of surface and less stable sperm proteins. Still, our dataset provided a robust proteome coverage and reproducibility.
Supplementary Information
Supplementary Material 1: Supplementary Datafile S1. TestSperm, CaputSperm and CaudaSperm total proteomes. Data related to Figure 1 and 2. Supplementary Material 2: Supplementary Figure S1. Classification of quantified proteins in TestSperm, CaputSperm and CaudaSperm proteomes performed in PANTHER. Numbers on the top of the bars represent the number of proteins associated with a specific protein class. Supplementary Material 3: Supplementary Figure S2. Localization of proteins in specific spermatozoa domains, as determined by* in silico* analysis. Quantified proteins in TestSperm, CaputSperm and CaudaSperm were mapped to specific sperm cell locations using Uniprot and EMBL-EBI GO annotations (retrieved in QUICKGO). Supplementary Material 4: Supplementary Datafile S2. Enrichment analysis of total proteomes and unique proteins from TestSperm, CaputSperm and CaudaSperm. Analysis performed using DAVID for Biological Process category considering an adjusted p-value (Benjamini) < 0.05. Data related to Figure 3 and 4. Supplementary Material 5: Supplementary Datafile S3. Analysis of Differentially Abundant Proteins (DAPs) between sperm populations. Upregulated (red) and downregulated (blue) between CaputSperm vs. TestSperm, CaudaSperm vs. TestSperm and CaudaSperm vs. CaputSperm. Enrichment analysis of upregulated and downregulated proteins was performed using DAVID considering an adjusted p-value (Benjamini) < 0.05. Data related to Figure 5. Supplementary Material 6: Supplementary Datafile S4. Analysis of proteins conserved among epididymal sperm proteomes of mammalian species (ram, pig and mouse). A total of 269 potentially conserved proteins were identified, and the respective enrichment analysis was performed using DAVID considering an adjusted p-value (Benjamini) < 0.05. Data related to Figure 7. Supplementary Material 7: Supplementary Datafile S5. Histones and histone variants identified in TestSperm, CaputSperm and CaudaSperm proteomes.
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