Proteomic Analysis of Capacitated and NonCapacitated Spermatozoa of Yanbian Yellow Cattle

Background: Sperm capacitation is a process which occurs prior to fertilization, and is essential for producing high-quality living embryos. The main purpose of this study was to explore the difference of proteomics between capacitated and non-capacitated sperm of Yanbian yellow cattle. Bioinformatic analyses of LC-MS/MS data included GO enrichment, KEGG pathway enrichment, and protein-protein interaction (PPI) analysis. Results: The results revealed 23 specic proteins in the capacitated group and 345 in the non-capacitated group. Compared with non-capacitated sperm, capacitated sperm exhibited 89 upregulated proteins and 509 downregulated proteins. Western blotting was used to conrm our proteomics data. The expression level of PSMD1 in the capacitated sperm group was signicantly lower than that in the non-capacitated sperm group, and the expression level of HSPA5 was signicantly higher than in the non-capacitated sperm group. Conclusions: Our results revealed that many proteins were differentially expressed between capacitated and non-capacitated sperm, particularly those involved in the proteasome signaling and protein transport signaling pathways. This work enhances our understanding of molecular processes involved in sperm viability in Yanbian yellow cattle, and provides a framework for future studies.

considered a protected national resource. Mass spectrometry is a comprehensive technique used in proteomics to elucidate protein levels and their composition in cells [9]. We used quantitative proteomics to study the differences in proteins of sperm in capacitated and non-capacitated states in Yanbian yellow cattle.
This study will add to the theoretical foundation needed for improving the breeding of Yanbian yellow cattle.

Semen collection and preparation
Three healthy and disease-free fresh semen from Yanbian yellow cattle aged 3-5 years were collected. The semen were washed with phosphate buffer saline (PBS) three times, sperm concentration was adjusted to 5 × 10 7 cells/mL, and centrifuged at 800 × g for 5 min to remove the seminal plasma. Then resuspended in noncapacitated medium (for non-capacitated treatment) and the capacitated medium (for capacitated treatment) respectively, both groups were incubated in 5% CO 2 incubator with maximum saturation humidity of 95% and a temperature of 38.5˚C for 1 h.

Capacitation status detection
The kinetic parameters of capacitated and non-capacitated sperm were measured by Computer Assisted Sperm Analysis (CASA) systems.
The tyrosine phosphorylation levels of bovine sperm protein were measured using western blotting. Sperm proteins were extracted from both the capacitated and non-capacitated groups, some sperm proteins concentrations were determined by the BCA kit, then SDS-PAGE and western blot analysis were performed.
Sperm acrosome morphological uorescence labeling staining According to the instructions in the peanut agglutinin uorescence labeling (PNA-FITC) staining kit (GENMED SCIENTIFICS INC.U.S.A), the sperm death/viability and acrosomal integrity were analyzed using uorescence microscopy and ow cytometry.

Filter Aided Proteome Preparation (FASP) enzymolysis
In each group, 100 µg of protein solution was made up to 100 µL with 25 mM ammonium bicarbonate. DTT (1 M) was added to the protein solution, which was then incubated at 57℃ for 1 h. Next, 10 µL of 1M iodoacetamide was added; the sample was mixed and incubated at room temperature for 40 min. Reductive alkylation proteins were ltered using a 10 K ultra ltration tube at 12,000 rpm, and discarded the solution at the bottom of the collection tube. Proteins bound to the membrane were washed four times with ammonium bicarbonate dissolution buffer. Trypsin formulated in dissolution buffer was added, and the proteins were digested overnight at 37℃. Enzymatically digested peptides were collected and concentrated by centrifugation, and dried.

ZipTip desalting
The dried polypeptides were concentrated by centrifugation, desalted on a ZipTip C18 column, dried, and prepared for mass spectrometry analysis. For desalting, peptides were reconstituted in 0.1% formic acid (FA). The desalting column was activated with 100% acetonitrile and equilibrated with 0.1% FA. Add the redissolved sample into the desalting column to make the sample ow slowly through the desalting column. Peptides trapped on the desalting column were washed with 0.1% FA solution and then eluted with a solution of 0.1% FA and 80% acetonitrile. The peptides were dried using a speed vacuum concentrator.

LC-MS/MS analysis
Vacuum dried samples were reconstituted with 0.1% FA, and 1-2 µg of peptide was used for LC-MS. Peptide separation was performed using an EASY-nLC 1200 (Thermo Scienti c, USA) equipped with a self-lled trap column (C18, 5 µm, 100 µm × 2 cm) and an analytical column (C18, 1.9 µm 75 µm × 20 cm) at a ow rate of 200 nL/min. Tandem mass spectrometry was performed on an Orbitrap Fusion Lumos (Thermo Scienti c, USA) in data dependent scan (Data Dependent Acquisition, DDA) mode. The full scan resolution was 60,000 (FWHM), and the mass-to-charge ratio range was set to m/z 375-1600. In HCD Fragmentation mode, the collision energy was set to 30%.

Quantitative analysis of protein
This experiment used the Precursor Intensity to conduct Label Free quantitative analysis. The iBAQ data and LFQ data in the MaxQuant software were used for analysis.

GO enrichment analysis
Gene Ontology (GO) annotates genes and gene products in terms of their cellular component, biological process, and molecular function. Enrichment analysis of KEGG pathway KEGG (Kyoto Encyclopedia of Genes and Genomes) is the main database used to systematically analyze information about gene functions, genomes and protein groups. When the pathway enrichment analysis was carried out, the different proteins were mapped with the pathways included in KEGG database. Furthermore, Fisher algorithm is used to calculate the probability of mapping differential proteins to different pathways, which is called pathway enrichment analysis. P < 0.05 indicating that the pathway was signi cantly enriched, which was affected by the experimental treatment.

Statistical analysis
All experiments were performed three times, and data were analyzed using Proteome Discoverer 1.4 and the Mascot server (Version 2.3, Matrix Science, London, UK). For western blot experiments, band density was determined using ImageJ software. All data were analyzed with ANOVA in using SPSS19. The Duncan test was used to compare the values and make multiple comparisons. When P < 0.05, the difference was considered to be signi cant ; and the test data were expressed as mean ± SD.

Detection of capacitation status
The kinetic parameters of sperm before and after capacitation are shown in Table 1. VCL (curvilinear velocity, m/s) and ALH (amplitude of lateral head displacement, μm) increased signi cantly after sperm capacitation (P<0.05), indicating that hyperactivation occurred during sperm capacitation.
The changes of protein tyrosine phosphorylation level are shown in Fig. 1. It can be seen from the gure that the protein tyrosine phosphorylation level of capacitated sperm is higher than that of non-capacitated sperm, indicating that protein tyrosine phosphorylation occurs during sperm capacitation.

Acrosome morphology test results
The results of uorescence microscopy ( Fig. 2) showed that the acrosome region of non-capacitated sperm showed bright green uorescence, and the uorescence of the capacitated sperm decreased slightly. Sperm in both treatment groups had strong acrosomal integrity, but a series of changes may occur in the acrosomal after capacitation, leading to a slight decrease in uorescence intensity.
The results of ow cytometry (Fig. 3) showed that the number of living cells decreased, the number of dead cells increased, and the integrity of acrosome and membrane decreased, which indicated that the membrane and acrosome were damaged during sperm capacitation.

Experimental enzyme digestion e ciency and search results
The enzymatic cleavage e ciency for each sample was counted using the MaxQuant software. The enzymatic cleavage e ciency of all samples was greater than 95% (Table 2). Table 3 shows the types of capacitated and non-capacitated proteins that were identi ed.

Qualitative analysis of proteins
The results of the detection of proteins which differed between the capacitated and non-capacitated sperm groups are shown in Table 4. Among the samples in the capacitation treatment group, 23 speci c proteins were identi ed. The non-capacitance treatment group had 345 speci c proteins. The analysis of protein similarities and differences between the two groups is shown in Fig. 4.

Protein Quantitative Analysis
Quantitative analysis was carried out for the protein whose normalized quantitative value was not 0 in all samples. In the inter group comparison, the normalized signal mean value of all samples in each group was calculated to calculate the inter group Ratio. The protein dataset was screened to determine differential proteins between groups using a cutoff of fold change ≥ 2 between groups (i.e., ratio ≥ 2 or ≤ 0.5) and P < 0.05. The proteins differentially expressed between the two groups are shown in Table 5. We identi ed 89 upregulated proteins and 509 downregulated proteins in the capacitation group compared to the noncapacitation group.

GO enrichment analysis
We next performed GO enrichment analysis to determine the functions of the genes differentially expressed between the capacitated and non-capacitated sperm. GO analysis is used to de ne the concepts related to gene functions and the way in which these functions are related. Analysis of cellular components for the 729 proteins indicated that nearly half of the differentially expressed proteins were located in "extracellular exosomes" and "extracellular spaces". The biological process analysis showed that almost all of the processes had many proteins that were downregulated in the capacitated sperm. In terms of molecular function, "cadherin binding involved in adhesion between cells" was the strongest classi cation (Fig. 5 A-C).

KEGG pathway enrichment analysis
To determine the pathways associated with the differentially expressed proteins, we performed KEGG pathway enrichment analysis (Fig. 6). Some of the important pathways are shown in Table 6. The analysis showed that metabolic pathways were the most involved, and that proteins such as SGSH, ARSB, and BTD are mainly involved in energy metabolism during sperm capacitation. In addition, we identi ed two other signaling pathways that appear to be important for sperm capacitation-proteasome signaling pathways and protein transport signaling pathways. PSMD1 and HSPA5 are essential proteins in both pathways respectively. The pathway map is shown in Fig. 7.
Protein-protein interaction (PPI) analysis PPI analysis was performed for each of the differentially expressed proteins. In the PPI interaction map, the node size indicates the number of proteins interacting with each other, with larger nodes indicating more protein interactions. The color of node indicates upregulation (red) and downregulation (green) , and the color intensity indicates the degree of modulation. The thickness of the edge indicates the reliability of the interaction,and the thicker the edge, the higher the reliability. The proteins involved in the proteasome and protein transport signaling pathways are shown in Fig. 8. These pathways are highly connected.

Western blot analysis
To validate the results obtained for differentially expressed proteins identi ed by quantitative proteomics, western blot analysis was used to examine the expression levels of the PSMD1 and HSPA5 proteins in capacitated and non-capacitated spermatozoa of Yanbian yellow cattle. As shown in Fig. 9 and Fig. 10, the expression levels of PSMD1 in the capacitated group was signi cantly lower than in non-capacitated group, and the expression level of HSPA5 in capacitated group was signi cantly higher than in the non-capacitated group. These results are consistent with our mass spectrometry results.

Discussion
After ejaculation, in order to acquire the ability to fertilize, sperm must undergo a maturation process called capacitation. It is generally believed that motility is one of the most important characteristics related to sperm fertilization, and it is an expression of sperm motility and structural integrity [10]. Hyperactivation is a highly asymmetric movement mode that occurs when the sperm is close to the oocyte. It is the guarantee for the sperm to complete the fertilization with the oocyte at the appropriate position and time. The increase of tyrosine phosphorylation (pY) is one of the most important processes in capacitation, protein phosphorylation in tyrosine residues has become a marker to de ne sperm capacitation [11]. This protein modi cation has been proposed to play a role in sperm motility in some species.

Changes of hyperactivation parameters before and after sperm capacitation
In most species, mature sperm remains immobile in the epididymis until released, and they quickly begin to swim. This process is called activation of motion. The capacitated sperm produces a nearly symmetrical agellum rhythm, which is driven by a nearly linear trajectory. When sperm are overactive, the extent to which the agellum bend is increased, usually on only one side of the agellum. Sperm hyperactivation is essential for fertilization because translucency of the zona pellucida requires sperm hyperactivation. A large number of studies in various species have shown that hyperactive exercise is essential for in vivo fertilization because it helps sperm penetrate the zona pellucida [12].
Computer Assisted Sperm Analysis (CASA) system has been used to detect the percentage of hyperactivated sperm. VCL is a representative index of hyperactive motility, VCL and ALH are parameters greatly increased in hyperactive sperm, BCF, ALH and VCL are indicative parameters of sperm motility [13]. The results showed that the VCL, ALH and BCF of capacitated sperm were higher than those of non capacitated sperm, noncapacitated spermatozoa were in a relatively low beat-cross frequency (BCF) and did not fully perform its swing function, it is suggested that the state of non capacitated sperm is to reduce energy consumption, so as to prepare for the increase of sperm swing frequency and smooth penetration of zona pellucida after capacitation [13]. LIN (linearity) represents the linear motion pattern of sperm. The LIN of the capacitated sperm was smaller than that of the non capacitated sperm, because the hyperactivation of the sperm after capacitation was highly asymmetric, and the linearity of the sperm movement decreased. STR (straightness) is a parameter of sperm motility. The STR of capacitated sperm is larger than that of non capacitated sperm, and the motility of capacitated sperm is enhanced, which is conducive to sperm penetrating the zona pellucida. The results showed that the LIN of the capacitated sperm was smaller than that of the non Page 8/30 capacitated sperm, and the STR of the capacitated sperm was larger than that of the non capacitated sperm. Studies have shown that sperm with high VSL (Straight line velocityhas) better sperm-oocyte binding ability [14], our results showed that the VSL of capacitated sperm was higher than that of non capacitated sperm.
Studies have shown that sperm without hyperactivation cannot cross the zona pellucida and bind to the oocyte [12]. Hyperactivation ability is regarded as an important index of male reproductive performance, and it is an indispensable link to ensure the successful fertilization of male animals [15]. It is known that the phosphorylation of sperm protein and the protein tyrosine phosphorylation downstream of the pathway are one of the maintenance conditions of sperm hyperactivity [16]. Therefore, the level of protein tyrosine phosphorylation was detected next.

Protein phosphorylation is a marker of capacitation
Post-translational modi cations, such as protein phosphorylation or dephosphorylation, regulate sperm capacitation, hyperactivation, and acrosome reactions, which are essential for sperm to reach, bind, penetrate, and fuse with oocytes [17,18]. In most species, immunocytochemistry has shown that tyrosine phosphorylated proteins are mainly located in the agellum of capacitated spermotozoa [19]. During capacitation, the distribution of tyrosine phosphorylation proteins in the agellum is related to sperm hyperactivation. The correlation between protein tyrosine phosphorylation and capacitation has not been fully established. However, some studies have shown that protein tyrosine phosphorylation is a marker of capacitation [20]. Arcelay and Visconti proposed that the increase of Tyr (tyrosine) phosphorylation level of sperm protein is usually considered as a marker of late capacitation [21,22]. Zapata et al. evaluated sperm capacitation by measuring protein tyrosine phosphorylation levels, and detected a signi cant increase in total phosphotyrosine content [23].
In mice, the presence of tyrosine phosphorylated proteins is considered essential for acrosome reactivity and fertilization in vitro, therefore it is regarded as the end point of capacitation. however some researchers believe that the appearance of tyrosine phosphoprotein is not always the end point of capacitation [24]. No fertilization has been reported in the absence of tyrosine phosphorylation. In this experiment, the curvilinear velocity and tyrosine phosphorylation level of capacitated sperm were signi cantly higher than those of noncapacitated sperm, indicating that hyperactivation and protein tyrosine phosphorylation occurred during sperm capacitation. Some studies have found that tyrosine phosphorylation is a marker of capacitation [25], leading to sperm hyperactivation and rapid beating of agella during asymmetric sperm movement. This result is consistent with this experiment. It has also been found that tyrosine phosphorylation proteins related to capacitation are associated with male fertility [26].
The role of the proteasome in sperm capacitation Proteasomes are responsible for the breakdown of most proteins in cells. The active site of proteolysis is located in the 20S core particles (CP). The only ATP-hydrolyzed proteins in the proteasome are the six ATPases present in the regulatory granules [27,28]. The main discovery with respect to proteasome regulation is that mammalian PKA phosphorylation stimulates the function of the 26S proteasome [29]. Proteasomes exist in sperm of many species and participate in a variety of sperm functions, including capacitation [30].
Several studies have shown that mammalian sperm have all of the components of the 26S proteasome.
A recent study described in detail the role of the proteasome degradation A kinase anchored brosheath protein AKAP3 (A kinase-anchored protein 3) in bovine sperm capacitation, this is related to the ability of the bull to acquire energy and acrosome exocytosis [31]. Bovine sperm shows activity of all three major proteasome core enzymes [32]. The acrosomal proteasome may play a dual role during the sperm-zona pellucida interaction, the rst is the mediator of acrosome exocytosis, followed by protease, which promotes the local degradation of zona pellucida matrix, leading to the formation of fertilization suture [33]. The degradation of soluble zona pellucida proteins by bovine sperm proteasome has not been reported, but it has been con rmed in humans [34] and Japanese quail [35] [36]. In humans, sperm proteasomes are involved in acrosomal exocytosis and the duration of Ca 2+ in ux before [37]. Sperm treatment with proteasome inhibitors blocks the ability and alters the pattern of protein phosphorylation without affecting sperm motility or ZP binding ability [38]. Furthermore, TK and PKA inhibitors decreased the activity of proteasome during capacitation. PSMD1 is a subunit of proteasome 19S regulatory granules and acts as a docking site for ADRM1, ADRM1 is another proteasome subunit that absorbs ubiquitinated substrates for proteolysis. PSMD1 is sumoylated by SUMO E3 enzyme PIASy. Ryu et al. found that sumoylation of a key lysine residue adjacent to the ADRM1 binding domain in PSMD1 regulates the association between ADRM1 and PSMD1. Moreover, the sumoylation-dependent interaction between PSMD1 and ADRM1 may alter the composition and function of the proteasome [39]. These ndings con rm the mechanism by which the SUMO family of ubiquitin-like proteins regulates ubiquitin-mediated protein degradation. Our results showed that the expression level of PSMD1 in the capacitated sperm group (H) of three Yanbian yellow cattle was signi cantly lower than that in the non-capacitated sperm group (F), indicating that ubiquitin mediated protein degradation may be involved in the capacitation process of bull sperm. Some reports suggest that ubiquitin proteasome system may play a role in sperm capacitation, acrosomal reaction, and/or sperm-oocyte fusion [40]. Pizarr et al. veri ed the existence of proteasomes in the sperms of several mammals, and Sanche et al. demonstrated their importance in the process of in vitro fertilization and acrosomal exocytosis in bovine [41]. All this evidence indicate that sperm proteasome plays an active role in the capacitation.

The role of chaperones in sperm capacitation
Chaperones are a group of proteins characterized by the ability to recognize the exposed hydrophobic surfaces of newly synthesized or partially folded proteins, and help them achieve their functional conformations. Thus, chaperone proteins prevent incorrect reactions that would otherwise lead to misfolding and aggregation of the protein. Prototypes chaperones were initially identi ed by increased expression after cells exposure to environmental stresses such as heat shock, and are therefore referred to as "heat shock proteins" (HSPs) or "cell stress response proteins". It is becoming increasingly apparent that these proteins play important roles in a variety of essential cellular processes that aid in protein folding, intracellular transport, membrane transport, and protein degradation. Therefore, these proteins are highly conserved during evolution, and are widely expressed in almost all eukaryotic and prokaryotic cells. Several somatic and germ cell-speci c molecular chaperones have been identi ed in male germ line, and appear to be essential for cell cycle progression in the initial stage of spermatogenesis [42]. Heat shock proteins are ubiquitous molecules in cells and acting as molecular chaperones under stress conditions, including carcinogenesis [43]. HSPA5 (GRP78) is involved in two signaling pathways: thyroid hormone synthesis and protein transport. Lachance et al. reported two separate chaperone proteins, HSP60 and GRP78, that are both expressed by oviduct epithelial cells (OECs) and regulated the potential for sperm motility, protein tyrosine phosphorylation, and intracellular calcium levels during capacitation. Our results showed that the expression level of HSPA5 in the capacitated sperm group (H) was signi cantly higher than that in the non-capacitated sperm group, suggesting that HSPA5 participates in the process of bull sperm capacitation. HSPA5 and HSPD1 proteins have been shown to bind to human sperm [44], and since chaperones are present in seminal plasma they may be adsorbed to the cell surface only after ejaculation and semen liquefaction [45]. Data from Lobo et al. also showed that GRP78 was phosphorylated at serine, threonine, and tyrosine residues, indicating that sperm GRP78 phosphorylation is converted during sperm maturation [46]. The heat-shock protein HSPA5 may be involved in capacitation.

Conclusion
In this study, we identi ed many differentially expressed proteins between capacitated and non-capacitated Yanbian yellow cattle sperm. The changes in PSMD1 and HSPA5 levels, which are associated with the proteasome signaling and protein transport signaling pathways. PSMD1 as a proteasome subunit regulates ubiquitin-mediated protein degradation, which may play a role in sperm capacitance in Yanbian yellow cattle. HSPA5, as a heat shock protein involved in protein transport pathway, may regulate sperm capacitation in Yanbian yellow cattle. These results provide a foundation for understanding the molecular processes driving sperm capacitation in these cattle, and for improving the reproductive capacity of Yanbian yellow cattle. However, this study is not deep enough, and further research is needed.    Figure 1 Phosphorylation levels of protein tyrosine in bovine spermatozoa of capacited and noncapacited Yanbian yellow cattle. The left gure shows the immunoblot result of the phosphorylation level of sperm protein tyrosine, and the right gure shows the relative expression levels.

Figure 1
Phosphorylation levels of protein tyrosine in bovine spermatozoa of capacited and noncapacited Yanbian yellow cattle. The left gure shows the immunoblot result of the phosphorylation level of sperm protein tyrosine, and the right gure shows the relative expression levels.
Page 18/30     indicates the trust degree of the interaction (the trust degree of the interaction relation is rated by the STRING database). The thicker the edge is, the higher the trust degree is. PPI interaction diagram The size of nodal protein represents the number of proteins interacting with it; The color of nodal protein indicates up-down, up-down to red and down to green; The darker the color is, the greater the modulation degree is. The thickness of the interaction relation line (edge) between node proteins indicates the trust degree of the interaction (the trust degree of the interaction relation is rated by the STRING database). The thicker the edge is, the higher the trust degree is. Western blot analysis of PSMD1 protein The result of western blotting is shown on the left, and the relative protein expression is shown on the right.

Figure 9
Western blot analysis of PSMD1 protein The result of western blotting is shown on the left, and the relative protein expression is shown on the right. Figure 10