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Proteomics and metabolomics analyses of mechanism underlying bovine sperm cryoinjury

BMC Genomics volume 26, Article number: 63 (2025) Cite this article

Abstract

Background

The cryoinjury of semen during cryopreservation reduces sperm motility, constraining the application of artificial insemination (AI) in bovine reproduction. Some fertility markers, related to sperm motility before and after freezing have been identified. However, little is known about the biological mechanism through which freezing reduces sperm motility. This study investigated the selective effects of cryoinjury on high-motility sperm (HMS) and low-motility sperm (LMS) in frozen-thawed from the perspectives of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and ATP levels. The molecular mechanism of decreased sperm motility caused by cryoinjury was explored through a joint analysis of 4D-label free quantitative proteomics and non-targeted metabolomics.

Results

The results indicate that low levels of ROS and high degrees of MMP and ATP play a critical role in the survival of HMS during the freezing process. The sperm samples from the frozen-thawed HMS and LMS were analysed for proteomics and metabolomics, 2,465 proteins and 4,135 metabolites were detected in bovine sperm samples. In contrast to LMS, HMS have 106 proteins and 106 metabolites with high abundance expression, and 79 proteins and 223 metabolites with low abundance expression. Proteomics and metabolomics data exhibit that highly expressed antioxidant enzymes and metabolites in HMS can maintain sperm motility by regulating the ROS produced during freezing to prevent sperm from oxidative stress and apoptosis. Furthermore, the KEGG analysis of differential proteins and metabolites during the freezing process implies that the significant enrichment of glycolysis and cAMP in HMS can guarantee energy supply.

Conclusions

The results provided that during the process of bovine sperm freezing, highly expressed antioxidant enzymes can regulate the reactive oxygen species levels to avoid oxidative stress and the glycolysis signalling pathway ensures ATP production can sustain frozen-thawed sperm motility.

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Introduction

AI technology can effectively promote bovine reproductive ability. Semen cryopreservation further ensures that AI is not limited by geography, time, and space. It can maximize the utilization efficiency and breeding rate of superior sires, possessing enormous economic value. However, the prominent problems of low sperm motility and short lifespan caused by cryoinjury after thawing have led to around 40–70% in bovine after AI [1, 2], reducing the efficiency of AI in bovine production. The frozen-thawed process of semen inevitably accompanies cryoinjury, which is attributed to extreme osmotic changes, cold shock, intracellular ice crystal formation, excessive production of ROS, and imbalance of the antioxidant defense system [3]. These processes ultimately induce the disruption of sperm morphology and physiological functions.

The decrease in sperm motility parameters is a significant indicator of the deterioration in the fertilization ability of sperm after cryopreservation [4]. However, these parameters cannot explain the molecular mechanism involved in the biochemical and physiological changes of sperm during the frozen-thawed process [5]. Mature sperm are terminal and highly differentiated cells without transcription and translation functions, coupled with their abundant, highly specialized, and partitioned characteristics, making proteomics analysis a useful approach [6, 7]. In the past decade, sperm proteomics analysis has gradually become a new strategy for searching for frost-resistance biomarkers of semen. Some studies compared the sperm of cows, goats, sheep, and horses before and after freezing using various proteomics techniques, and multiple protein markers related to motility and frost resistance were identified [8,9,10,11]. The dynamic relationship between proteins and metabolites allows the biological system to function as a cohesive unit. Metabolites are indispensable in the biochemical environment as they are the primary components of all protein biochemical structures [12]. Metabolomics is a key scientific field in the post-genomic era, which investigates small molecules to supplement genomics, transcriptomics, and proteomics and helps to identify new disease biomarkers and treatment strategies [13]. Metabolomics analysis has been applied in semen cryoinjury in yak, sheep, and pig to determine metabolic markers relevant to frost resistance in sperm or seminal plasma [14,15,16]. However, most studies merely focus on valuable markers related to the viability of frozen semen, with little attention to the mechanism underlying sperm death during freezing. The main focus of this investigation is the cause of the simultaneous appearance of live and dead sperm in the same semen sample when frozen under the same circumstances. Therefore, the current authors consider that it is essential to analyze the cryoinjury mechanism by examining the differences between live and dead sperm in frozen-thawed semen. The Percoll gradient centrifugation method with different concentrations can separate live and dead sperm from frozen-thawed semen, with LMS and HMS as the characteristics of the low-density group and the high-density group, respectively [17]. Reports on the mechanism of semen cryoinjury using this method are only concerned with yak and cow. They identified multiple markers related to sperm motility through the proteomics analysis of HMS and LMS separated from frozen-thawed semen [17, 18]. However, further explorations of the biological mechanism underlying this phenomenon during semen freezing are not conclusive.

Based on the above facts, this study evaluated the damaging effect of freezing on sperm by measuring the motion parameters, intracellular ROS concentration, MMP, and ATP concentration of HMS and LMS isolated from bovine frozen-thawed sperm. The mechanism of cryoinjury affecting sperm motility was disclosed using 4D-label free quantitative proteomics and untargeted metabolomics.

Results

Percoll separation of frozen-thawed sperm

After thawing, the semen was placed on the upper layer of 90 − 45% Percoll and underwent centrifuge to separate HMS and LMS. CASA was used to evaluate the motion parameters of separated sperm. Compared with the LMS collected at the 45–90% interface, all motion parameters indicators collected at the 90% interface in HMS were superior (P < 0.05) (Table 1). Through principal component analysis (PCA), sperm dynamics parameters were simplified into two variables to reflect velocity and linearity, respectively (Fig. 1A). The principal component diagram depicts that the samples are well separated (Fig. 1B). The significant difference in HMS and LMS motion parameters provides a basis for the accuracy of proteomics and metabolomics analysis.

Table 1 The motion parameters of HMS and LMS separated by Percoll from bovine frozen-thawed semen
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Data are expressed as the mean ± standard error of the mean. VCL: curvilinear velocity, VSL: straight line velocity, VAP: average path velocity, BCF: beat-cross frequency, ALH: amplitude of lateral head displacement, STR: straightness (VSL/VAP), LIN: linearity (VSL/VCL), WOB: wobble (VAP/VCL).

Fig. 1
figure 1

PCA of sperm motility and motion parameters. (A) Sperm motility and motion parameters are depicted as vectors according to PC1 and PC2. The direction of these vectors is determined by the component loading; (B) The distribution of individuals based on PC1 and PC2. Individual points are grouped in ellipses

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The relationship between the motility of frozen-thawed sperm and ROS

The results of flow cytometry analysis show that the ROS content in HMS is evidently lower than that in LMS (Fig. 2A) (P < 0.05), and the motility of frozen sperm has a negative correlation with ROS (Table S1).

The effect of frozen sperm motility on mitochondrial membrane potential and ATP levels

The fluorescence of MMP is shown in Fig. 2C. Red/orange represents high potential, and green denotes low potential. According to flow cytometry analysis, the MMP in HMS is significantly higher than that in LMS (P < 0.05), indicating a positive correlation between mitochondrial activity and sperm motility (Table S1). ATP is crucial for maintaining sperm motility and movement. The results exhibit that ATP in HMS is profoundly higher than in LSM (Fig. 2B) (P < 0.05), implying a positive correlation between ATP and sperm motility (Table S1).

Fig. 2
figure 2

Physiological parameters of HMS and LMS isolated from bovine frozen-thawed sperm. (A) Detection of ROS in sperm using the DCFH-DA probe; analysis of the peak value of ROS and monitoring the fluorescence intensity in sperm with flow cytometry; (B) The concentration of ATP in sperm; (C) After staining sperm with JC-1, mitochondrial activity is detected by loss cell assay, with red/orange representing high potential and green indicating low potential

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Proteomics analysis

Global proteomics changes in bovine sperm cryoinjury

This study described how changes in bovine sperm motility caused by deep-frozen affect protein abundance. A total of 17,707 peptide segments and 2,465 proteins are identified in bovine sperm, of which 2,403 were quantified (97.4%) (Table S2). The quality control analysis of the proteome was also assessed including peptide lengths (Figure S1A) and peptide distributions (Figure S1B). Moreover, PCA was used to investigate protein expression patterns. The results demonstrate that HMS and LMS samples have good clustering performance (Figure S1C), representing that the motility of frozen-thawed semen has a significant impact on protein expression patterns. Next, this study combined multiple changes in abundance (greater than 1.5) and the P-value < 0.05 to compare the abundance of HMS and LMS proteins. In contrast to LMS, HMS has 106 proteins with high abundance expression and 79 proteins with low abundance expression (Fig. 3A Table S3). Hierarchical cluster analysis was also conducted for DEPs, the result of which were illustrated by a heat map (Fig. 3B), this demonstrated changes in proteomes of HMS and LMS.

Functional analysis of differentially expressed proteins

To determine the potential biological role of DEPs in frozen-thawed bovine sperm with high and low motility, functional classification was performed on these proteins using multiple bioinformatics analysis methods. According to the three classifications of GO (biological process, cellular component, and molecular function), functional analysis was conducted on DEPs. (1) Biological process: Upregulated proteins mainly involve the metabolic process, glycolysis process, fertilization, and cell redox homeostasis. Downregulated proteins are related to protein folding and response to endoplasmic reticulum stress. (2) Cellular composition: Upregulated proteins concern cytoplasm, cytosol, and cilium. Downregulated proteins involve cytoplasm, endoplasmic reticulum, and supramolecular complex (such as endoplasmic reticulum lumen, endoplasmic reticulum subcompartment, and endoplasmic reticulum membrane), indicating a correlation between endoplasmic reticulum and sperm motility. (3) Molecular function: Upregulated proteins are mainly relevant to antioxidant activity, kinase activity, and oxidoreductase activity. Downregulated proteins primarily involve purine ribonucleoside triphosphate binding and protein folding chaperones (Fig. 3C, D, Table S4, 5).

Based on the KEGG database, KEGG enrichment pathway analysis was performed on the DEPs of HMS and LMS in bovine frozen-thawed sperm to obtain potential signalling pathways. In this study, upregulated proteins are significantly enriched in 31 signalling pathways, including metabolism, glycolysis/gluconeogenesis, and the PPAR signalling pathway (Fig. 3E, Table S6). Downregulated proteins are significantly enriched in 16 signalling pathways, involving the protein processing in endoplasmic reticulum, Ras signalling pathway, and apoptosis (Fig. 3F, Table S7).

Fig. 3
figure 3

Identification and functional enrichment analysis of DEPs between HMS and LMS in bovine frozen-thawed sperm. (A) Volcano plots demonstrating the DEPs between HMS and LMS; (B) Cluster analysis of DEPs identified between HMS and LMS; (C and D) GO analysis of upregulated and downregulated proteins between HMS and LMS based on biological processes, cellular components, and molecular functions; (E and F) KEGG pathway analysis of upregulated and downregulated proteins between HMS and LMS

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GSEA analysis

Since GO and KEGG enrichment analyses of signalling pathways require screening for DEPs first, which is based on a specific degree of fold change and significance analysis, the biological results have some limitations. Therefore, the GSEA enrichment analysis was further conducted. Different from Go and KEGG analyses, enrichment analysis is performed on all expressed proteins and determines the effects of some gene sets on biological processes based on protein expression levels. Meanwhile, it can predict whether enriched signalling pathways are activated or inhibited in biological processes. Therefore, GSEA enrichment analysis was adopted in this study on all quantitative proteins to validate signalling pathways that changed in the motility-related proteome in frozen-thawed semen. The results of the GSEA analysis reveal that 12 gene sets related to signalling pathways are upregulated in HMS, including the activation of metabolic pathways, glycolysis/gluconeogenesis, and the cAMP signalling pathway gene sets. In LMS, seven gene sets related to signalling pathways are upregulated, mainly involving the activation of the gene set of apoptosis (Fig. 4, Table S8).

Fig. 4
figure 4

GSEA plots for proteomics results based on KEGG pathways. (A) The Glycolysis/Gluconeogenesis associated hallmark gene set is upregulated in HMS; (B) The cAMP signalling pathway associated hallmark gene set is upregulated in LMS; (C) The apoptosis-associated hallmark gene set is upregulated in HMS. Green curves correspond to enrichment score (ES) curves, representing the running sum of weighted ES values derived from the GSEA software. Normalized enrichment score (NES) are used in graphs

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PPI analysis

To investigate the interactions between DEPs of HMS and LMS separated from bovine frozen-thawed sperm and their involvement in the cross-linking of various biological networks, PPI analysis was performed on the DEPs using a string database. The results show that the interactions between DEPs present high complexity and are closely linked. Compared to LMS, most proteins are highly expressed in HMS. Differential proteins identified were classified based on known biological functions. It is found that they mainly participate in metabolic processes and proteolysis, which play a vital role in fertilization. Among them, proteins with more interaction nodes contain PARK7, PGK1 and PRDX6, representing their significant effect on regulating the motility of frozen-thawed bovine sperm (Fig. 5).

Fig. 5
figure 5

The predicted PPI network for DEPs between HMS and LMS samples. In the constructed network, DEPs are represented by nodes, while interactions among proteins are denoted by edges for proteins with more than two connections. Significantly upregulated and downregulated proteins are marked in red and green

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Western blot validation

To verify the results of the above quantitative proteomics analysis, this study selected two proteins PARK7 and TPPP2, and identified the abundance of their HMS and LMS isolated from frozen-thawed sperm through the western immunoblotting method. The results disclose that PARK7 and TPPP2 have higher abundance in HMS, which agrees with the results of 4D proteomics analysis (Fig. 6A), implying that the proteomics data in this study are accurate and reliable.

Immunofluorescence localization of PARK7 and TPPP2

The immunofluorescence method was adopted to observe the positions of PARK7 and TPPP2 proteins in HMS and LMS separated from bovine frozen-thawed sperm. PARK7 is mainly located in the posterior region of the sperm head in HMS and the acrosome and the posterior region of the sperm head in LMS, with a significantly diminished expression level. TPPP2 is located in the acrosome and flagella in HMS, while in LMS, it only exists in the acrosome, and its expression level remarkably drops (Fig. 6B). These results indicate that the motility of bovine frozen-thawed sperm is related to the expression level and localization of PARK7 and TPPP2.

Fig. 6
figure 6

Western immunoblotting and Immunofluorescence. (A) Western immunoblotting-based validation of quantitative proteomics results for two representative proteins, with α-tubulin as a loading control. (B) Immunofluorescence localization results of PARK7 and TPPP2 in bovine HMS and LMS. Green represents Glycoprotein immunolocalization by the specific antibody TRITC. Blue denotes sperm nucleus stained by DAPI. Merge represents the superposition effect of the fluorescence of the two channels. The scale bar is 50 μm

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Metabolomics analysis

Identification and classification of metabolites

To reveal the molecular mechanism of sperm motility diminution during freezing, untargeted metabolomics was used to investigate the metabolic differences between HMS and LMS isolated from bovine frozen-thawed sperm. A total of 4,135 metabolites are identified, of which 2,484 are in the positive ion mode and 1,651 are in the negative ion mode (Table S9). The OPLS-DA analysis was carried out on HMS and LMS samples to eliminate irrelevant differences and differentiate between them. As shown in the OPLS-DA scoring table (Figure S2A, Figure S2B), samples in the same group are relatively clustered, and samples from different groups are evidently dispersed, denoting good repeatability within the same group and metabolic differences between groups. To evaluate the predictability and reliability of the OPLS-DA model, seven cross-validation and 200 response ranking tests were conducted. The regression line of Q2 is always lower than that of R2, and its intercept with the y-axis is less than zero (Figure S2C, Figure S2D), indicating that the model is reliable and there is no overfitting. Therefore, the obtained VIP values can be used to screen for DEMs.

This study conducted a pooled analysis of positive and negative ion patterns. Different levels of metabolites may contribute to the changes in sperm motility after freezing and thawing. A total of 329 DEMs are identified in HMS, composed of 106 upregulated and 223 downregulated (Fig. 7A, Table S10), mainly including benzene and substituted derivatives, carboxylic acids and derivatives, fatty acyls, glycerophospholipids, organooxygen compounds, prenol lipids, and steroids and steroid derivatives. The cluster analysis was employed to explore the accumulation of DEMs in sperm. The results present that after sperm freezing, the concentration of most metabolites elevates with sperm motility diminution (Fig. 7B), demonstrating that sperm cryoinjury produces more metabolites.

KEGG analysis of sperm metabolites

The metabolites differentially expressed in HME and LMS are significantly enriched in the KEGG pathway. KEGG analysis shows that 25 signalling pathways are significantly enriched in upregulated DEMs, mainly involving the cAMP s signalling pathway, mTOR signalling pathway, and pyruvate metabolism (Fig. 7C, Table S11). Eleven signalling pathways are significantly enriched in downregulated metabolites, mainly related to metabolic pathways (Fig. 7D, Table S12).

Fig. 7
figure 7

Metabolomics analysis of DEMs between HMS and LMS. (A) Volcano plot of DEMs between HMS and LMS. (B) Hierarchical clustering heat map of DEMs between HMS and the LMS. The colour bar and the number in the upper right corner represent the relative abundance of metabolites. Red indicates that the metabolite is upregulated, and blue denotes the downregulated metabolite. (C) KEGG pathway analysis of upregulated metabolites between HMS and LMS. (D) KEGG pathway analysis of downregulated metabolites between HMS and LMS

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Integrated analysis of proteomics and metabolomics

This study summarized the potential challenges during the process of freezing and thawing of bovine sperm using proteomics and metabolomics data combined with physiological indicators (Fig. 8). The freezing and thawing process generates superfluous ROS, leading to severe oxidative stress in the sperm. Correspondingly, oxidative stress affects mitochondrial integrity and energy production, which mainly occurs in the middle of the sperm and damages sperm motility. Autophagy is subsequently triggered, resulting in sperm apoptosis. In addition, the inhibition of glycolysis (the ATP production pathway) and cAMP is another reason for insufficient energy and reduced motility during sperm freezing (Fig. 8).

Fig. 8
figure 8

Cellular response in bovine sperm after frozen-thawed. The frozen-thawed process decreases ATP generation by glycolysis and cAMP signalling pathways, which in turn impair progressive motility. The frozen-thawed process damages the antioxidant system to produce abundant ROS, inducing severe oxidative stress in the sperm to impair motility. In response to oxidative stress, autophagy is triggered for protection but eventually guides the sperm to step into apoptosis as cellular damage is severe

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Discussion

Semen cryopreservation can cause increased fluidity and permeability of the sperm plasma membrane, downgraded acrosome integrity, abnormal flagella, mitochondrial damage, and oxidative stress induced by increased reactive oxygen species. These changes will alter the structure of lipids and proteins, lower sperm motility, and exacerbate sperm DNA fragmentation, resulting in declined quality and fertilization rate of frozen-thawed sperm [19,20,21]. Current studies on the mechanism of semen cryoinjury mostly focus on the biological changes between fresh sperm and frozen-thawed sperm and promote the quality of frozen-thawed sperm by adding antioxidant-active substances [22]. However, during the process of freezing and thawing, surviving spermatozoa undergo prominent modifications, including their structure and physiological status [23, 24]. Therefore, the authors believe that investigating surviving and dead sperm after the frozen-thawed process is valuable for improving sperm quality, as they are subjected to the same freezing process. By analyzing the motion parameters of HMS and LMS isolated from 90 − 45% Percoll in bovine frozen-thawed sperm, this study found that all indicators in HMS were significantly higher than those in LMS, such as total mobility, progressive mobility, VCL, VSL, and VAP. It confirms that this study can separate surviving and dead sperm during the freezing and thawing process through Percoll, providing reliable support for probing the biological mechanism of bovine semen cryoinjury. Since fertility is a multi-parameter process, a single parameter of sperm quality is insufficient to evaluate the overall fertility potential of semen samples. Some potential protein and metabolite biomarkers about motility decrease cryopreservation sperm were identified in multiple mammalian species such as ram and boar [25, 26]. Therefore, the proteomics and metabolomics are a promising strategy for identifying potential biomarkers of bovine sperm motility decrease caused by cryoinjury and understanding the respective biologic functions.

Identification and analysis of proteins and metabolites in bovine sperm cryoinjury

Applying the 4D-label-free technology, this study identified and quantified 2,403 proteins in bovine sperm, of which 106 were upregulated in HMS and 79 were downregulated. Their differences may be a factor for sperm motility decreased. The results represent that some proteins with antioxidant capacity, such as PARK7 and PRDX6, are highly expressed in HMS. They can retain sperm vitality by clearing ROS produced during sperm freezing. Moreover, metabolic pathways related to energy metabolism, especially proteins involved in glycolysis, are highly expressed in HMS. They can generate ATP in sperm through metabolism without producing ROS, which meets the energy needs of sperm while preventing oxidative stress [27]. Therefore, this study speculates that the loss of proteins that maintain these functions during the freezing process of bovine sperm may induce sperm motility diminution. It is worth noting that signalling pathways of spermatogenesis and structural reorganization, such as those related to chaperone complexes and protein folding, are specifically identified in bovine sperm and negatively correlated with fertilization rate [28]. The endoplasmic reticulum plays a crucial role in the folding and assembly of newly synthesized proteins in mammalian cells. However, it is eliminated during spermatogenesis. A highly active protein synthesis and folding event occurs before spermatogenesis is completed [29]. Therefore, this study believes that the activation of protein folding and endoplasmic reticulum pathways in LMS may reflect abnormal protein folding during sperm freezing, ultimately affecting the low viability of frozen-thawed bovine sperm.

Through the untargeted metabolomics method, this study identified 4,135 metabolites in bovine sperm, of which 106 were upregulated in HMS and 223 were downregulated. These are the most identified metabolites in bovine sperm so far, laying the foundation for future research on the metabolic function of bovine sperm [30, 31]. DEMs analysis reveals that metabolites related to the cAMP signalling pathway and pyruvate metabolism are highly expressed in HMS, indicating that metabolites and proteins in sperm interact with metabolic pathways to generate ATP and sustain sperm energy supply. Fan et al. discovered that adding galactose during freezing semen could increase ATP levels by augmenting AKR1B1 protein expression, thereby enhancing frozen-thawed sperm motility [14]. This indicates that combining proteomics with metabolomics to investigate the disequilibrium of metabolic pathways in bovine sperm cryoinjury is essential to adjust the sperm ATP synthesis pathway.

ROS generated during the frozen-thawed process induces oxidative stress in sperm

The imbalance between the antioxidant defense system and ROS production in sperm cells during cryopreservation leads to oxidative stress [32]. This study speculated that oxidative stress during the frozen-thawed process of bovine sperm might be the main cause of sperm motility diminution. To verify this assumption, this study detected the ROS content in HMS and LMS isolated from bovine frozen-thawed sperm. The results exhibit that the high content of ROS in LMS greatly impairs sperm motility. Meanwhile, this study conducted proteomics analysis on HMS and LMS. It is found that proteins related to oxidative stress (PRDX6 and PARK7) are significantly downregulated in LMS. The reduction in the expression of these proteins indicates that the antioxidant system has been damaged in LMS, producing more ROS. Shi et al. reported similar findings when simulating oxidative stress by supplementing exogenous H2O2 to sperm and freezing semen [21]. PARK7 mainly serves as an oxidation-reduction sensitive partner and oxidative stress sensor to respond to cellular damage caused by oxidative stress [33]. The level of PARK7 in human sperm is positively correlated with the integrity, vitality, and SOD activity of the sperm plasma membrane [34]. PARK7 can protect cells from oxidative stress by clearing ROS through autoxidation [35]. Although it has been reported that PARK7 can affect the viability of yak sperm after freezing and thawing, the potential changes in the localization of PARK7 during mammalian sperm freezing are discussed for the first time. The immunofluorescence analysis shows that the localization of PARK7 in HMS differs from that in LMS after sperm freezing. In HMS, it is mainly located in the posterior half of the head, while in LMS, it is located in the acrosome and posterior half of the head. This suggests that the migration of proteins during sperm freezing may affect motility. Additionally, PARK7 has been localized on both human and pig flagella. However, in this study, it was not expressed on bovine flagella, indicating species specificity in the localization of PARK7 in sperm [36, 37]. The sensitivity of the plasma membrane of sperm to oxidative stress is attributed to their composition of unsaturated fatty acids [38]. In this study, metabolites linked with oxidative stress, such as L-homocitrulline, acetylcarnitine, and Isobutyryl-l-carnitine, are significantly downregulated in LMS. It has been reported that adding citrulline and carnitine to the semen freezing extender can enhance the antioxidant capacity and mitochondrial membrane potential of bovine and sheep sperm, improving semen quality [39,40,41].

Therefore, the results of this study suggest that the decrease in levels of key antioxidant proteins and metabolites leads to oxidative stress which effect the motility after cryopreservation of sperm.

Cryopreservation can stimulate autophagy of sperm mitochondria

Cellular autophagy is a conservative self-degradation that occurs in various stress responses, such as oxidative stress and heat stress. Pamela Uribe et al. found that exposure to oxidative stress could activate autophagy in human sperm, thereby preventing impaired sperm motility and cell death [42]. However, the latest studies put forward that mitochondrial autophagy is significantly activated in frozen-thawed sperm. Although autophagy can help sperm cope with mild oxidative stress caused by ROS, when severe ROS-induced damage is caused by sperm freezing, autophagy leads to programmed cell death of sperm. Given the high degree of cytoplasmic degradation of sperm, autophagy mainly occurs in mitochondria [21]. In this study, proteins related to mitochondrial autophagy (NGF and CLU) are significantly upregulated in LMS, and LMS presents severe oxidative stress and MMP damage. Furthermore, according to a recent study, adding H2O2 to human sperm to simulate oxidative stress and the occurrence of oxidative stress during frozen-thawed both cause sperm autophagy and apoptosis, resulting in decreased sperm motility [21]. Therefore, we assumed that oxidative stress-induced mitochondrial autophagy during the freezing process of bovine sperm plays a crucial role in viability decrease. Autophagy can facilitate, conflict, or cooperate with other cell death processes, including apoptosis and necrosis, serving either a pro-survival or pro-death function [43]. KEGG analysis exhibits that the apoptotic signalling pathway is significantly enriched in LMS. The GSEA enrichment analysis of all expressed proteins in HMS and LMS sperm reveals that the apoptotic signalling pathway is activated in LMS. Therefore, it is suspected that oxidative stress during sperm freezing can stimulate significant autophagy in sperm, leading to programmed cell death of sperm through the apoptotic signalling pathway. It agrees with previous studies on cell apoptosis induced by autophagy [21, 44]. Induced autophagy seems to help sperm cope with mild oxidative stress caused by ROS. However, in the case of severe ROS-induced damage during sperm freezing, autophagy may lead to programmed cell death of sperm.

Cryopreservation can change ATP production in sperm

Sperm movement is generated by flagellar movement with high-energy consumption after ATP hydrolysis [45]. This study found that HMS isolated from frozen-thawed sperm had higher mitochondrial membrane potential and ATP, indicating that reduced ATP production caused by the impaired mitochondrial function of LMS is the primary factor in sperm motility diminution. Thoroughly analyzing this mechanism through molecular biology methods is particularly important for improving semen cryoinjury.

The ATP required for bovine sperm to maintain vitality is produced jointly by glycolysis and oxidative phosphorylation, and the former is dominant [46, 47]. The proteomics analysis in this study reveals that the glycolysis signalling pathway is solely significantly enriched in HMS. The significant downregulation of glycolysis enzymes GPI, ENO3, PGAM2, FBP1, GALM, and PGK1 in LMS may be the major reason for the inhibition of ATP production in the glycolysis pathway, which corresponds to the low level of ATP in LMS. In addition, this study conducted GSEA enrichment analysis on all expressed proteins of HMS and LMS. It was found that the glycolysis signalling pathway was activated in HMS. It demonstrates that the results of bioinformatics analysis in this study are reliable and can provide solid support for subsequent biological mechanism research. PGK is the main enzyme involved in ATP production in the glycolysis pathway. It can convert 1,3-bisphosphoglycerate and ADP into 3-phosphoglycerate and ATP. It has been verified that PGK2 is a key protein affecting sperm motility and fertility [48]. In this study, the expression level of PGK1 in HMS is evidently higher than that in LMS. Therefore, it is rational to assume that the high level of PGK1 after sperm freezing affects sperm motility by regulating ATP production through the glycolysis pathway. PGAM2 is a catalytic enzyme that promotes the conversion of 3-phosphoglycerate to 2-phosphoglycerate in the glycolysis pathway and participates in other metabolic processes by balancing the conversion between 3-phosphoglycerate and 2-phosphoglycerate [49, 50]. The prominent drop of PGAM2 in LMS can be considered as the inhibition and effect of ATP production by the glycolysis pathway to influence sperm motility. It has been reported that adding cholesterol-loaded cyclodextrin to the freezing extender of sperm can abate the degradation of PGAM2 in frozen-thawed sperm, minimize the impact of cryopreservation on glycolysis, and enhance sperm motility [49]. Therefore, we speculate that the downregulation of these glycolysis enzymes effect of ATP production, which can affect sperm motility during cryopreservation. But specific mechanisms which need further research.

ATP and adenosine are two derivatives of purine, playing crucial roles in maintaining cellular energy balance and nucleotide synthesis, respectively [51]. Purine and adenosine receptors can intervene in the regulation of cAMP levels to enhance sperm function and vitality [52]. The metabolomics results of this study demonstrate that purine metabolism and the cAMP signalling pathway are significantly enriched in HMS. Metabolites associated with them, including acetylcholine, adenosine 5’-monophosphate, adenosine monophosphate, and d-myo-inositol-1,4,5-triphosphate, are overexpressed in HMS, which is essential for cAMP to maintain a high level to ensure high sperm motility during semen frozen-thawed. A study has found that the changes in cAMP within sperm cells are consistent with ATP production. Adding exogenous cAMP to the semen-freezing extender can promote sperm motility after freezing and thawing [53, 54]. It indicates that the cAMP signalling pathway can prevent sperm motility diminution during semen frozen-thawed by retaining ATP concentration, which is in accordance with the higher levels of ATP in HMS sperm. In addition, the GSEA analysis of sperm proteomics in this study shows that cAMP is activated in HMS, which can activate PKA and increase tyrosine phosphorylation to maintain sperm mitochondrial function and ATP production [55]. The phosphorylation of flagella protein through the cAMP/PKA signalling pathway can boost sperm motility [56]. TPPP2 is a mitochondrial function-related protein specifically expressed in the reproductive organs of male animals. When TPPP2 is inhibited in human and mouse sperm, its vitality and ATP content significantly abate. Furthermore, after knockout TPPP2 in mice, sperm motility and ATP content significantly decreased, accompanied by a significant drop in sperm count and mitochondrial structure damage [57]. In this study, TPPP2 is significantly downregulated in the LMS isolated from frozen-thawed sperm. To verify the function of TPPP2 in bovine sperm frozen-thawed, this study conducted the immunofluorescence experiment. The results present that TPPP2 is located in the flagella in HMS and the acrosome in LMS. It implies that changes in the localization of TPPP2 during semen freezing may involve impaired mitochondrial function, resulting in decreased ATP synthesis. The expression level of TPPP2 may thus be potential biomarker of sperm motility. This is the first time that TPPP2 has been localized in mature mammalian sperm, laying a valuable foundation for studying TPPP2 sperm function.

Conclusion

In summary, cryopreservation divides bovine sperm into HMS and LMS groups. On the whole, highly expressed antioxidant enzymes in HMS can sustain sperm motility by regulating the ROS produced during freezing to avoid oxidative stress and apoptosis. The glycolysis pathway in HMS ensures ATP production which can maintain sperm motility. The key proteins, metabolites and pathway identified in this research provides new insights into the molecular regulatory mechanism of sperm cryoinjury during cryopreservation and the improvement of frozen semen motility.

Materials and methods

Reagent

All chemicals not specified were purchased from Sigma-Aldrich (MO, USA).

Semen cryopreservation

Fresh semen was obtained from four Gaoqing bulls (4–6 years) at the Aohang farm in Shandong Province, China, using an artificial vagina. To evaluate the progressive motility of sperm, 3 μL of fresh semen was immediately evaluated through a computer-assisted sperm analysis system (CASA, Nikon, Eclipse E200, Basler, acA780-75gc, SCA sperm class analyzer). Only semen samples were considered for analysis with a volume ≥ 2.0 mL and revealed ≥ 70% motility. A Biladyl® extender from Minitube, GER was employed to mix the samples in 2 steps. In the first step, the glycerol-free solution was mixed with the samples, yielding 50% of the total volume. They were then stored for 2 h at 5 °C. Secondly, the samples were again mixed with a chilled (5 ºC) solution containing glycerol and then stored with the same condition. The sperm were then stored (cryopreserved) with a final 140 to 200 × 106/mL cell density. The cooling curve was implemented as follows: -5 ºC/min from 5 to 4 ºC, -3 ºC/min from 4 to -10 ºC, -40 ºC/min from − 10 to -100 ºC, and − 20 ºC/min from − 100 to -140 ºC. All samples were stored in liquid nitrogen for long-term storage.

Sperm sample preparation

After thawing at 37ºC for 30 s, sperm samples were added in 1.5 mL of 45% Percoll and 1.5 mL of 90% Percoll in a 15 mL conical plastic tube. To separate HMS and LMS, sperm suspensions were spread on the upper gradient layer and spun for 10 min at 700 × g to distinguish between HMS and LMS. The abnormal morphology of spermatozoa, and seminal extender were recovered from the top layer of 45% Percoll; LMS was observed from the 45–90% Percoll interface; HMS was recovered from the bottom layer of 90% Percoll yielded HMS. Sperm quality parameters were evaluated using CASA as per the World Health Organization standards [58].

Spermatozoon ROS detection

The levels of ROS production in spermatozoa were measured via the ROS Kit and DCFH-DA ROS probes, in line with the manufacturer’s recommendation. The sperm (10 × 106/ mL) and DCFH-DA (10 μM) were incubated in a dark condition for 30 min at 37 ºC. DCFH-DA was transformed into fluorescent 2,7-dichlorofluorescein within the cell due to the presence of intracellular ROS. The intensity of fluorescence was quantified via flow cytometry.

Spermatozoon of MMP detection

Sperm mitochondrial activity was detected via the JC-1 dye under the provided protocols. Approximately, 100 μL of the sperm (10 × 106/mL) were mixed with JC-1 staining solution (10 μL) and kept in a dark condition at 37 ºC for 30 min. The sample was spun at 4 ºC, 600 × g for 5 min, followed by the 1 mL addition of JC-1 staining buffer. The sample was again centrifuged at the same conditions. Afterwards, the sample was diluted properly with 200 μL of JC-1 dye. The fluorescence intensity of JC-1 was determined via flow cytometry.

Spermatozoon ATP measurement

The phosphomolybdic acid colorimetric method was used to evaluate ATP concentration in sperm as per the provided instructions via an ATP detection kit. Approximately lysis buffer (200 μL) was mixed with the sperm sample (10 × 106mL) and kept for 30 min on ice. The suspension concentration was estimated by collecting it after 10 min of spinning at 12,000 × g and 4 ºC. The sample was boiled in the water bath for 10 min and consistently mixed in the ATP stock solution (reagent kit) as the reference solution. After 5 min of sample incubation at ambient temperature (20–25 ºC), the absorbance was measured on a 96-well plate using a multifunctional microplate reader. Based on a linear correlation between absorbance and ATP concentration, the ATP levels of the sperm were quantified.

Quantitative proteomics analysis

Protein extraction and trypsin treatment

There were 100 million sperm in each sample. HMS and LMS samples were washed twice with PBS for 10 min at 2,000 × g and mixed in a lysis buffer containing SDS (1%), a protease inhibitor (1%), TSA (3 μM), and NAM (50 mM). These homogenates three times using were sonicated (thrice) via a high-intensity ultrasonic processor (Scientz, Ningbo, China). The samples were spun for 10 min at 12,000 × g, 4 ºC to obtain supernatant. Protein was quantified in the supernatant via a BCA reagent (Beyotime Biotechnology, Wuhan, China) following the manual’s recommendations. Enzymolysis was carried out for each protein sample (100 μg), followed by mixing it with the same volume of lysis solution. The sample was then mixed with one volume of chilled acetone, vigorously mixed, and then diluted with four volumes of chilled acetone. Afterwards, it precipitated for 2 h at -20 °C, then dissolved again in TEAB (200 mM), and distributed using ultrasonication. First, the samples of protein were digested by incubating them with trypsin at 1:50 (protein and trypsin) for 24 h. The sample was treated with DTT to obtain a 5 mM concentration and reduced to 56 ºC for 30 min. It was adjusted to 11 mM by adding IAA, and the sample was then kept in the dark at 20–25 ºC for15 min.

LC-MS/MS analyses

All peptides were added and dissolved in a solution containing formic acid (0.1%) and acetonitrile (2%), and then promptly loaded onto a reverse-phase analytical column. The solvent B (formic acid (0.1%) in acetonitrile) concentration was progressively increased from 9 to 24% > 5 min, from 24 to 35% > 3 min, and from 35 to 80% > 4 min to perform gradient elution. The samples were immersed in solvent B (80%) for 4 min with a flow rate of 450 nL/min via an EASY-nLC 1200 UPLC system (Thermo Scientific, T CA, USA). Afterwards, the peptides were examined via the Capillary source on the timsTOF Pro mass spectrometer (Thermo Scientific) that was plugged online into the UPLC system, followed by MS/MS. The voltage of the ion source was adjusted to 1.75 kV. The peptide precursor ions and secondary fragments were discovered and examined using TOF. Data-independent parallel accumulation serial fragmentation (dia-PASEF) mode was implemented for data collection. The initial range for scanning in mass spectrometry (MS) was fixed at 100–1700 m/z. PASEF mode collections were carried out ten times after the acquisition of a single primary MS image. In the secondary MS scanning, the window was every 25 m/z, with a range of 400–1200.

Database searches

In this study, the obtained MS/MS spectra were compared to those in the UniProt database (Bos taurus, sequences: 37,508) via concatenated reverse-decoy database searching with the Maxquant search engine (v.1.8). The cleavage enzyme was designated as trypsin/P, and a maximum of one cleavage was accepted. The fixed change was carbamidomethyl on Cys, while the variable changes were Met oxidation, deamidation (NQ), N-terminal acetylation, and phosphorylation on Thr, Ser, and Tyr. The value of FDR was adjusted at ≤ 1%.

Statistical analysis

Data was statistically evaluated via the SPSS Statistics software (IBM, NY, USA, version 28.0). The correlation between motility parameters and ROS, MMP, ATP were assessed by Pearson correlation (data normally distributed). A fold-change cut-off of 1.5 and a p ≤ 0.05 were used to recognize differentially expressed proteins (DEPs). Sperm treatments were compared using a t-test. Data were illustrated as the mean ± SEM. Significant and highly substantial variances were depicted by p-values of ≤ 0.05 and 0.01, respectively.

Bioinformatics analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses of the DEPs recognized between HMS and LMS spermatozoa and conducted via the KOBAS (http://kobas.cbi.pku.edu.cn/annotate/) and g: Profiler (https://biit.cs.ut.ee/gprofiler/gost) tools. Significantly enriched pathways were identified as those with a corrected p ≤ 0.05 The gene set enrichment analysis (GSEA v 4.1.0) evaluated the protein enrichment in samples within previously recognized pathways [59]. Significantly enriched pathways were identified based on an FDR-adjusted q ≤ 0.25 and NOM p ≤ 0.05. The STRING (v11.5) database (https://string-db.org/) was used to conduct a screening of established protein-protein interactions (PPIs) which was created via Cytoscape software (v 3.9.1), excluding networks with ≥ three nodes [60].

LC/MS-based metabolomics analysis

Metabolite extraction

The sperm sample (10 × 106) were washed twice with PBS for 10 min at 2,000 × g and vortexed with 1 mL of pre-cooled acetonitrile: carbonitrile: aqueous suspension (2:2:1, v/v) for 30 s. The solution was kept at a low- temperature for ultrasound exposure for 30 min and stewed at -20 ºC for 10 min. This mixture was spun at 14,000 g and 4 ºC for 20 min and the supernatant containing metabolites was vacuum desiccated. An acetonitrile aqueous suspension (100 μL) (H2O: acetonitrile, = 1:1, v/v) was added to the sample, vortexed, and spun at 14,000 g and 4 ºC for 15 min to conduct MS analysis. The supernatant was preserved for additional analysis. The QC sample was prepared by extracting 10 μL from each sample to assess the LC-MS system’s accuracy and reproducibility.

LC-MS/MS analysis of metabolite samples

Metabolites were separated via an advanced UPLC (Agilent 1290 Infinity LC) with a HILIC column. The mobile phase A consists of C2H7NO2 (25 mM) and NH4OH (25 mM). The flow rate was adjusted at 0.5 mL/min with 2 μL of injection volume. The mobile phase B was NH4OH. In the UPLIC gradient, B was adjusted at 40% for 0–9 min, and it varied linearly from 40 to 95% for 9–9.1 min. From 9.1 to 12 min, B was sustained at 95%. The sample was adjusted at 4 ºC in an autosampler during the whole analysis. Continuous analysis of samples was conducted randomly to remove the effect of signal fluctuations on detection. The system stability and the accuracy of experimental data were monitored and evaluated by incorporating the QC samples into the sample queue.

The samples’ primary and secondary spectra were acquired in real-time via the AB Triple TOF 6600 mass spectrometer, which runs in a dual (+ ve/-ve) ESI turning mode. The total scanning time was 0.20 s/spectrum, and the m/z ratio measurement range in primary MS was 60-1000 Da. The m/z ratio in secondary MS was between 25 and 1000 Da, with an average detection time of 0.05/spectra. In secondary MS, the m/z ratio varied between 25 and 1000 Da, with an overall scanning period of 0.05/spectra. The secondary MS was carried out using information-dependent acquisition (IDA). The screening was conducted in the peak intensity mode. The declustering potential was defined as ± 60 V, and 35 ± 15 eV was the collision energy. The IDA configuration included a dynamic exclusion range of 4 Da for isotopic ions, and each scan acquired 10 fragment spectra.

Metabolomics data analysis and path enrichment

Proteo Wizard was employed to change the raw data into mz XML format. The retention time correction, peak alignment, and area extraction were implemented using XCMS software. The metabolite integrity of the data derived from XCMS was verified, and metabolites with missing values ≥ 50% were excluded. To ensure parallelism in comparing metabolites and samples, the total peak area of the data was normalized. The OPLS-DA model determined the variable importance in projection (VIP). The paired Student’s t-test estimated the p-value in a single-dimensional statistical analysis. Metabolites that revealed a p ≤ 0.05, FC ≥ 1.2 (or FC ≤ 0.83), and VIP ≥ 1.0 were classified as highly differential expressed metabolites (DEMs) [61]. The biological pathways of DEMs were examined via the KEGG pathway database.

Western blotting analysis

Bovine sperm proteins were loaded on 12% SDS-PAGE via electrophoresis after denaturation of the 5X SDS loading buffer. These proteins were transferred to the PVDF membrane which was blocked with skim milk (5%) at 22–25 ºC for 2.5 h. After this, they were added Rabbit polyclonal anti-α-tubulin (1:2000; Proteintech11224-1-AP, China), rabbit polyclonal anti-PARK7 (1:1000, abcom18257, UK), and rabbit polyclonal anti-TPPP2 (1:1000; abcom236887, UK) and incubated overnight at 4ºC. After TBST stripping, ). Goat polyclonal Rabbit IgG secondary antibody (1:2000, Bioss-2405R, China) was added also after TBST stripping. The protein bands were captured by the CCD camera system (Tanon, Shanghai, China) and visualized using the ECL chemiluminescence assay reagent. ImageJ (National Institutes of Health, NIH) was employed to analyze all images. The results of two target proteins were standardized using β-actin as the internal reference.

Immunofluorescence

The sperm sample was preserved in paraformaldehyde (4% PFA) for 15 min and approximately 10 uL sample was dropped onto a coated glass slide. The slide was then kept in an oven at 37 ºC for 30 min. The sample was permeablized with 0.5% Triton-100 for 10 min, followed by blocking with horse serum (10%) for 1 h. Primary antibodies (1:400) PARK7 and TPPP2 were then added, and the sample was kept at 4 ºC overnight in a moist slide box. Next, a secondary antibody: goat anti-rabbit Alexa Fluor® 488 IgG H&L (1:500, abcom150077, UK) was added to the slide, and incubated for 1 h at 20–25 ºC in a dark condition. All nuclei of the spermatozoa were stained via DAPI (10 μg/mL) (Solarbio-C0065). After the addition of an anti-fluorescence quencher and mounting the slide, the smear was examined via an ultra-high resolution fluorescence microscope (ZEISS, Scope. A1).

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) with the data set identifier PXD049265, the mass spectrometry metabolomics data have been deposited to the Open Archive for Miscellaneous (https://ngdc.cncb.ac.cn/omix) with data set identifier OMIX005798.The data supporting the conclusions of this study are available in the supplementary table and figure.

Abbreviations

AI:

Artificial insemination

CASA:

Computer-assisted sperm analysis system

DEMs:

Differentially expressed metabolites

DEPs:

Differentially expressed proteins

GO:

Gene Ontology

GSEA:

Gene set enrichment analysis

HMS:

High-motility sperm

KEGG:

Kyoto encyclopedia of genes and genomes

LMS:

Low-motility sperm

MMP:

Mitochondrial membrane potential

PCA:

Principal component analysis

ROS:

Reactive oxygen species

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Authors and Affiliations

  1. College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, 266109, China

    Renzheng Zhang, Xiuyuan Wang, Ruili Liu, Yanfang Mei, Xiuping Miao, Jiaxu Ma, Lei Zou, Qiuyue Zhao, Xuejin Bai & Yajuan Dong

  2. Laboratory of Animal Molecular Shandong Black Cattle Breeding Engineering Technology Center, Qingdao, 266109, China

    Renzheng Zhang, Ruili Liu, Xuejin Bai & Yajuan Dong

Authors
  1. Renzheng Zhang
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  2. Xiuyuan Wang
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  3. Ruili Liu
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  4. Yanfang Mei
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  10. Yajuan Dong
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Contributions

R.Z., Y.D. and X.B. conceptualized this study. X.W and R.L. helped in the investigation. Y.M. and X.M. helped in methodology and software. J.M., L.Z. and Q.Z. performed data curation. R.Z. wrote the original draft. R.L and Y.D. helped in writing, reviewing, and editing the manuscript. Y.D. helped in funding acquisition. All authors read and approved the manuscript.

Corresponding author

Correspondence to Yajuan Dong.

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Ethics approval and consent to participate

All animals were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People’s Republic of China, and the study was approved by The Animal Administration and Ethics Committee of Qingdao Agricultural University, Animal Science and Technology College (Permit No. 20230610).

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Not applicable.

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The authors declare no competing interests.

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Zhang, R., Wang, X., Liu, R. et al. Proteomics and metabolomics analyses of mechanism underlying bovine sperm cryoinjury. BMC Genomics 26, 63 (2025). https://doi.org/10.1186/s12864-025-11258-w

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  • DOI: https://doi.org/10.1186/s12864-025-11258-w

Keywords

BMC Genomics

ISSN: 1471-2164

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