LCAT acts as a molecular switch and regulates rooster sperm motility in vitro

Abstract

Sperm motility is a critical determinant of semen quality, yet the molecular mechanisms regulating motility remain unclear. In this study, we identified Yufen 1 roosters with significant differences in sperm motility among highly motile sperm samples stored under different conditions. By integrating the seminal plasma proteome and seminal plasma metabolome data, 181 proteins and 112 metabolites were identified as differentially expressed between the high motility group (HMG) and low motility group (LMG). Lecithin cholesterol acyltransferase (LCAT) was upregulated in the LMG. Targeted LCAT activation significantly reduced sperm motility and plasma membrane integrity, caused sperm plasma membrane disruption, chromatin loss, and mitochondrial swelling, and reduced the longest fertilization days and number of fertilized eggs. Mechanistically, LCAT regulates sperm motility in vitro by altering sperm membrane structure and function by modulating glycerophospholipid metabolism. These findings provide new insights into extending the window period for artificial insemination and semen preservation strategies in poultry.

Graphical Abstract

Highlights

• •Seminal plasma modulates rooster sperm survival, motility and membrane stability.
• •Seminal PC-cholesterol exchange remodels sperm membranes to tune in vitro motility.
• •Seminal LCAT varies with rooster fertility, proving seminal proteins govern fertility.

In Brief

By integrating seminal plasma proteome and seminal plasma metabolome data, LCAT regulates sperm motility in vitro by regulating glycerophosphatidylcholine metabolism and changing sperm membrane structure and function. Targeted LCAT activation significantly reduced sperm motility and plasma membrane integrity, caused sperm plasma membrane disruption, chromatin loss and mitochondrial swelling, and shortened the maximum fertilization days and the number of fertilized eggs. This will provide new insights into the strategy of extending the AI window period and semen preservation in poultry.

Fecundity is an important indicator of optimal performance in poultry production, and males produce highly motile sperm (1). Sperm motility is a key factor in male fertility (2) and a key predictor of semen quality, fertilization potential, and sperm preservation (3). Thus, sperm motility is an important indicator of rooster fecundity. Seminal plasma (SP) is a complex biological fluid and has an important impact on sperm cell survival and function (4). SP can enhance sperm motility and significantly affect the sperm preservation time in vitro in poultry (5). SP proteins are significantly correlated with semen quality and fertilization rate (5, 6, 7). Poultry semen typically has a high sperm concentration, which results in rapid changes in sperm metabolism and function (5). The interaction between SP and sperm induces metabolic changes that alter sperm membrane structure and function (8).

The plasma membrane is an important structural component of living cells and serves as a special barrier separating the internal and external environments, enabling the isolation of intracellular organelles (9, 10). Plasma membrane lipids play a crucial role in various physiological and pathological processes by maintaining the structure and function of organelles (9, 11). The four main types of plasma membrane lipids are glycerophospholipids, sphingolipids, glycolipids, and sterols (12). The fluidity and flexibility of the cell membrane mainly depend on its lipid composition (13). Sperm membrane integrity and fluidity are important for sperm motility, capacitation, the acrosome reaction (AR), and sperm-egg fusion (14). Sperm membrane components, especially lipids, are directly involved in core sperm functions, including membrane fluidity, sperm motility, and sperm-oocyte binding (13, 15). The fatty acid composition of sperm plasma membrane is directly related to sperm motility (16, 17). Cholesterol is an important component of the sperm membrane, which is important for regulating membrane fluidity. The cholesterol level is significantly higher in the sperm membrane of infertile patients than in that of fertile males (18).

In recent years, proteomics has become an effective technique for evaluating the effects of animal SP on fecundity (7), and important seminal plasma proteins associated with semen quality have been analyzed in various animals. For example, PSP-I in boar SP is associated with low sperm motility (19), FAM115 in rabbit SP is associated with sperm membrane integrity (20), and SPINK2 plays an important role in the maintenance of chicken sperm motility (21). In addition, potential protein and metabolite biomarkers associated with sperm motility preservation have been reported in humans (22), mice (23), cattle (24), and sheep (25). Therefore, SP proteins play a crucial role in regulating male semen quality.

Metabolites are the end products of metabolic pathways and play important roles in sperm physiology, such as motility, energy metabolism, and metabolic regulation (26). Metabolites in SP, such as amino acids, peptides, sugars, fatty acids, steroids, and nucleosides, are involved in important physiological activities that affect sperm energy production, motility, pH, membrane protection, and metabolic activity (27, 28). Metabolomics is a key scientific field in the post-genomic era that studies small molecules to complement genomics, transcriptomics, and proteomics and helps identify new disease biomarkers and therapeutic strategies (29). Metabolomic analyses have identified metabolic markers associated with sperm motility in the sperm and SP of yaks (30), sheep (31), and pigs (32). Metabolites are the main components that make up the biochemical structure of all proteins (33). Dynamic relationships between proteins and metabolites allow biological systems to function as cohesive units. Thus, SP proteomics and metabolomics are effective strategies for identifying biomarkers that affect sperm motility and understanding their respective biological functions. Of particular interest in regulating sperm motility is the fact that avian sperm motility occurs mainly after ejaculation, and sperms with equally high motility can produce significant differences in motility under the same conditions. Therefore, molecular switches controlling motility activation and inactivation in response to extracellular stimuli may operate in avian sperm. However, the mechanisms underlying avian fertilization, particularly in terms of sperm function and mechanism, have not yet been elucidated.

Experimental Procedures

Ethics Approval

All experimental protocols in this study strictly followed the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Henan Agricultural University (approval number 19-0068).

Design of the Trial

Proteomic and metabolomic analyses were performed to explore the possible roles of key SP proteins and metabolites in maintaining sperm motility (Fig. 1A). The effect of SP on sperm motility was evaluated by removing and cross-exchanging SP. In addition, the co-culture of SP with sperm helped investigate the effect of these metabolites on motility preservation. Paired experiments were performed in a crossover fashion for incubation tests. All indices were assessed by repeating the test three times and averaging.

Fig. 1.

Experimental scheme of the study and phenotypic determination. Experimental scheme of the study (A). Body weight (B), sperm motility (C), membrane integrity (D), longest fertilization days (E), and number of fertilized eggs (F) between the HMG and LMG. Mean values with different letters differ significantly at p < 0.05 (Student’s t test, n = 10 or 100).

Experimental Design and Statistical Rationale

One hundred and 47 individuals with high sperm motility (≥0.8) were selected from 530 healthy breeding roosters of the same generation. 20 individuals with the same extreme difference in sperm motility were selected after preservation at 4 °C and 37 °C, which were defined as the high motility group (HMG motility ≥0.6) and the low motility group (LMG motility ≤0.1), and 10 fresh sperm were collected from each group. After centrifugation at 3000 rpm for 5 min, the upper seminal plasma was aspirated into a 1.5 ml enzyme-free tube and flash frozen in liquid nitrogen and stored at −80 for biochemical indicators detection and omics analysis. Three biological replicates of the high and low groups were used for proteomics sequencing analysis, and six biological replicates of the high and low groups were used for metabolomics sequencing analysis.

Statistical analysis of omics data was mainly performed by R software (version 4.0), and proteomics was performed by fold change (fold change, fold change). Proteins with FC ≥1.2 or ≤0.83 (absolute log2FC ≥0.263) and p < 0.05 (| log2FC | ≤0.263 & p < 0.05) were used as the threshold for screening differentially expressed proteins. In metabolomics, the metabolite data were filtered (more than 80% of the sample was missing, or more than 50% of the QC was missing), data imputation (KNN method was used by default), and data standardization (PQN method was used by default) to obtain the final expression value. The metabolites that met the three conditions of p Value <0.05 from t test, |log2 FC| >0.263 and VIP ≥ 1 calculated by PLSDA analysis were the final significant differential metabolites.

Experimental Animals

The experimental animals were obtained from the Poultry Germplasm Resource Farm of Henan Agricultural University. A model for sperm motility selection for ex vivo semen preservation was established using HMG and LMG. First, individuals with sperm motility >0.8 were selected, and semen from these individuals was diluted and stored at 37 °C for 2 h and 4 °C for 5 days, after which, individuals showing extreme differences under the two conditions were selected (high sperm motility >0.5 and low sperm motility ≤0.1). These individuals were artificially inseminated into the same batch of hens, and the longest fertile egg days (DN) and fertile egg number (FN) were used to evaluate the survival rate of sperm in the oviduct of hens after a single artificial insemination (34, 35). Subsequently, the same individuals with extreme differences were selected again as HMG and LMG models for further experiments. Individuals in the model were healthy roosters of the same generation with a uniform appearance. The diet was prepared to meet the nutritional requirements of Yufen1 chickens (Supplemental Table S1).

Sample Collection

Fresh semen from HMG and LMG was collected into semen collection centrifuge tubes and centrifuged at 3000 rpm for 10 min at 4 °C. The resulting supernatant, representing the SP, was collected in a 1.5 ml tube. Part of this sample was used for phenotypic index analysis, and the remaining part was collected into frozen tubes in liquid nitrogen and frozen at −80 °C for proteomics and metabolomics sequencing analysis.

Seminal Plasma Clearance and Cross-Exchange Assays

Sperm and seminal plasma were separated from fresh HMG and LMG semen by centrifugation. The effect of seminal plasma on sperm function was evaluated using seminal plasma removal and cross-exchange tests. The hypotonic swelling test (HOST) (36) and gentian violet staining (37, 38) were used to detect sperm membrane integrity and malformation rate, respectively.

Hatching Trial

To analyze the maintenance of vitality in high- and low-level spermatozoa in vivo, semen with varying vitality levels selected at different temperatures were artificially inseminated after density adjustment. Each rooster was paired with 10 hens, and high- and low-viability sperm were exchanged for insemination. Fertilized eggs were collected for 20 days, starting from the third day post-insemination, and labeled. There was a 30-days interval between each batch of inseminated fertilized eggs. The same batch was incubated at a constant temperature (37.8 °C, relative humidity 50%) for 7 days, and the eggs were candled to record fertilized and unfertilized eggs; dead embryos were considered as fertilized eggs. The sperm survival rate and survival time in the reproductive tract of hens were tested using the longest fertilization egg days (DN, the number of days from insemination to the production of the last fertilized egg) and the number of fertilized eggs (FN, the number of fertilized eggs following insemination) (34, 35, 39).

Sperm Membrane Integrity Analysis

A hypoosmotic swelling test was performed to assess the functional membrane integrity of sperm according to a previously described method (36). A hypoosmotic solution (157 mOsm/L) containing 7.35 g/L of sodium citrate and 13.51 g/L of fructose was incubated with semen (1:10, v/v) at 35 °C for 60 min. After incubation, 10 μl of the mixture was placed on a pre-warmed slide under a coverslip and observed under 400 × magnification for counting the frequency of sperm tails swelling.

Sperm Malformation Rate Determination

Sperm morphology abnormalities were evaluated using gentian violet staining (37, 38). Briefly, semen samples were diluted 1:100 with pre-warmed physiological saline (37 °C), thoroughly mixed, and 10 μl aliquots were smeared onto slides. After fixation with anhydrous ethanol (2–3 min), slides were stained with 10 μl gentian violet solution (3 min) and air-dried. A total of 200 spermatozoa per sample were examined at 400 × magnification to quantify the morphological defects.

Membrane Transmission Electron Microscopy of Sperm

Sperm samples were fixed using 1 ml of 0.5% glutaraldehyde and 4% paraformaldehyde to preserve membrane structures. Subsequently, 1% osmium tetroxide was used to stabilize lipids and enhance membrane contrast. Sperm samples were progressively dehydrated using acetone and embedded in Epon-812. Ultrathin sections (90 nm) were collected on copper grids, double-stained with uranyl acetate for 15 min and lead citrate for 2 min and imaged using a JEM-1400FLASH transmission electron microscope.

Seminal plasma Proteomics

For each sample, 500 ng of total peptides were separated and analyzed with a nano-UPLC (Vanquish neo) coupled to an Astral instrument (Thermo Scientific) with a nano-electrospray ion source. Separation was performed using a reversed-phase column (EASY-Spray HPLC(150 um × 15 cm),Thermo Scientific, USA). Mobile phases were H₂O with 0.1% FA (phase A) and 80% ACN with 0.1% FA (phase B). Separation of sample was executed with a 6.9 min gradient. Data independent acquisition (DIA) was performed in profile and positive mode with Orbitrap analyzer at a resolution of 240K and m/z range of 380 to 980 for MS1; For MS2 m/z range of 150 to 2000. The HCD with normalized collision energy (NCE) of 25% and an isolation window of 2 m/z.

A data-independent acquisition (DIA) data acquisition method was used. That is, based on the mass-to-charge ratio (m/z), the entire scanning range of the mass spectrum was divided into several windows. Then, all the parent ions in each window were fragmented and detected, and the fragment ion information of all parent ions was collected for qualitative and quantitative protein analysis. Protein search identification and quantitative analysis of the DIA mass spectrometry data were performed using DIA-NN software (https://www.nature.com/articles/s41592-019-0638-x) (version 1.9.2). Key DIA-NN parameters: Protein identification mode set to DirectDIA (eliminating the need for actual DDA data to construct a spectral library; Instead, a theoretical virtual spectral library is built directly using the protein FASTA sequence database from the corresponding species in the UniProt database. The FASTA file was downloaded on May 13, 2024, and contains 51,562 proteins). Enzyme digestion parameters set to Trypsin/P, with a maximum missed cleavage count of 2. Fixed modifications: Carbamidomethylation (C). Variable modifications: Oxidation (M), Acetylation (Protein N-terminal). Peptide length range: 7 to 37. Precursor m/z range: 380 to 980. Fragment ion m/z range was set to 150 to 2000, Quantities matrices set FDR threshold to 0.01, Peptidoforms, MBR, isoform IDs, and protein identification confidence strategy were set to Target-decoy, Protein identification threshold was set to spectrum false positive (PSM FDR) < 0.01, Protein FDR <0.01. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD073221. (URL Link: https://www.iprox.cn/page/PSV023.html;?url=1768809265342q5l1, password: xQXy).

Seminal Plasma Metabolomics

SP samples were thawed on ice, and the metabolites were extracted using a 50% methanol buffer. Chromatography: An ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm, Waters) was used for separation. The mobile phase consists of phase A (5 mmol/L ammonium acetate + 5 mmol/L acetic acid + water) and phase B (acetonitrile). Gradient elution conditions were set as follows: 0∼0.8 min, 2% ∼ 70%B; 0.8∼2.8 min, 70% ∼ 90% B; 2.8∼5.3 min, 90% ∼ 99% B; 5.3∼5.9 min, 99% B; 5.9∼7.5 min, 99% ∼ 2% B;7.5∼7.6 min, 2% B;7.6∼10.0 min, 2% B; The flow rate is 0.3 ml/min. The injection volume for each sample was 4 μl. The column oven was maintained at 40 °C.

Mass spectrometry: A high-resolution tandem mass spectrometer Q-Exactive Plus (Thermo Fisher Scientific) was used to detect metabolites eluted form the column. Each sample was operated in both positive and negative electrospray ionization mode. ESI temperature is 350 °C. The voltage is +3800 V in positive ion mode and −3400 V in negative ion mode. The sweep gas pressure of the ion source is 0 Arb, Gas 1 (Auxiliary gas) pressures set to 15 Arb, Gas 2 (Sheath gas) pressures set to 50 Arb. The mass spectrometric data were obtained with full scan and data-dependent acquisition (DDA) modes. In one acquisition cycle, the full scan acquisition range is 70 to 1050 Da, and the resolution is 70k, AGC target is set to 3E6,Maximum IT is set to 100 ms. Then, the top five signal ions with a signal accumulation intensity of more than 100,000 were selected from the full scan for DDA scanning, the DDA resolution is 17.5k, Maximum IT is set to 50 ms. Dynamic exclusion is set to 6 s.

Convert LC-MS raw data files into mzXML format, then process them using XCMS software in R. Identify ions by combining retention time (RT) and m/z data. Record the intensity of each peak and generate a three-dimensional matrix containing any specified peak indices (RT-m/z pairs), sample names (observables), and ion intensity information (variables). Metabolites were identified using online databases (MoNA, GNPS, HMDB) and an in-house constructed database. Metabolic ions were identified as metabolites when the mass difference between observed and database values was less than 10 ppm and the match score exceeded 70. Metabolite annotation was performed using KEGG and HMDB databases.

Statistical analysis was primarily performed using R software (version 4.0). Metabolite data underwent filtering (excluding samples with >80% missing values or >50% missing QC data), data imputation (default KNN method), and data normalization (default PQN method) to obtain final expression values. Metabolites meeting all three criteria—P value < 0.05 from t-tests, fold change |log2 FC| > 0.263, and VIP ≥ 1 from PLSDA analysis—were identified as significantly differentially expressed metabolites (40).

Analysis of the key Molecules in SP

SP concentrations of phosphatidylcholine (PC), lysophosphatidylcholine (LPC), lecithin-cholesterol acyltransferase (LCAT), and free cholesterol (FC) levels were quantified using commercial ELISA kits (Jiangsu Enzyme Immunoassay). Absorbance at 450 nm was measured using a microplate reader, and sample concentrations were determined using standard curves according to the manufacturer’s guidelines.

Targeted LCAT Activation in Combination With Sperm Incubation

The LCAT agonist Apolipoprotein A1(APOA-I) was used at concentrations of 0.01 mg/ml, 0.10 mg/ml, and 1.00 mg/ml. NaCl was used as the control. HMG sperm were added to each group at 10%, and sperm motility was measured. The 1.00 mg/ml concentration produced the most significant difference and was selected as the optimal concentration for subsequent experiments. Subsequently, sperm deformity rate and membrane integrity were assessed. A sperm concentration was standardized, and 50 μl of sperm was inseminated into each hen, with five hens per group. Eggs were collected daily for 20 days and incubated at a constant temperature. DN and FN levels were calculated to determine the effect of the targeted activation of LCAT on the maintenance of sperm motility in the reproductive tract of hens.

Statistical Analysis

Data are presented as means and standard deviations. Data were checked for normality and equal variance using the Shapiro-Wilk and Levene’s tests, respectively. When the data were normally distributed and exhibited homogeneity of variance, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test or Student’s t test was performed to test for differences between the groups. p ≤ 0.05 was considered statistically significant, and 0.05 < p < 0.1 was considered a tendency.

Results

Sperm Properties

Sperm samples from 40-week-old Yufen1 breeder roosters of similar body weights were divided into LMG and HMG (Fig. 1, B and C); the groups showed significant individual differences in sperm viability (p < 0.05). After adjusting sperm density through 1:1 dilution of equally motile samples and storing at both 4 °C and 37 °C, the high-motility sperm maintained superior viability at both temperatures (Fig. 1C). At varying temperatures, low-motility sperm exhibited significantly lower (p < 0.05) membrane integrity than HMG (Fig. 1D). Compared with the LMG, hens that received high-motility sperm presented significantly greater (p < 0.05) longest fertilization days and number of fertilized eggs (Fig. 1, E and F).

Seminal Plasma Composition Drives Sperm Function

To evaluate the role of SP in sperm function, spermatozoa and seminal plasma were separated by centrifugation and subjected to SP removal and cross-exchange SP tests (Fig. 2A). When the SP was removed from the semen, the motility, malformation rate, and membrane integrity of sperm were comparable between the HMG and the LMG (Fig. 2, B–D). However, the cross-exchange of SP between the HMG and the LMG resulted in decreased sperm motility (p < 0.05) and membrane integrity (p = 0.053), along with a similar malformation rate (Fig. 2, E–G). These results indicate that SP composition remarkably influences sperm function and survival in Yufen1 roosters.

Fig. 2.

The influence of seminal plasma on sperm parameters. Experimental scheme of seminal plasma (SP) removal and exchange SP test (A). The effects of SP removal (B–D) and SP exchange (E–G) on sperm motility, malformation rate, and membrane integrity. Mean values with different letters differ significantly at p < 0.05 (Student’s t test, n = 10).

Metabolite Changes in Response to Sperm Motility

A metabolomic analysis was performed to determine the relationship between SP composition and sperm motility. The metabolomic composition of the HMG was different from that of the LMG, with a clear separation visualized in the plots of the partial least-squares discriminant analysis (PLS-DA) and principal component analysis (PCoA) (Fig. 3, A and B). A total of 1578 DMs, 555 upregulated and 1023 downregulated metabolites (Fig. 3C), were identified in terms of primary mass spectrometry (MS1) metabolites (p < 0.05). Furthermore, 31 and 77 metabolites were upregulated and downregulated, respectively, in the two-stage mass spectrometry metabolites (MS2) (Fig. 3D). The significantly altered metabolites included 3-methylheptanedioylcarnitine, LPC, phosphatidylethanolamine (PE), phosphatidylserine (PS), PC, proline, lysophosphatidylethanolamine (LPE), and lysine. Of these, PC and LPC were the primary differentially abundant metabolites (Fig. 3E). KEGG and GSEA analyses further revealed that these DMs were enriched in 118 pathways, with the most pronounced differences occurring in the glycerophospholipid metabolism pathway, followed by lysine degradation and arachidonic acid metabolism (Fig. 3, G–I). The differentially abundant metabolites in the glycerophospholipid metabolism pathway included PE, PS, PC, and PLC. In the arachidonic acid metabolism pathway, the key protein was L-PGDS, and the differentially abundant metabolites included PC and 5(S)-HETE. Metabolites in the efferocytosis pathway primarily include LPC, PS, and L-arginine. PE is the main metabolite of autophagy in animals. The main protein and metabolite involved in ferroptosis were PRNP and glutamate, respectively (Supplemental Table S2). Quantitative analysis revealed that SP from high-motility sperm exhibited significantly decreased LCAT levels (p < 0.05), the key enzyme catalyzing PC-to-LPC conversion, increased PC and free cholesterol (FC) levels, and reduced PC content (Fig. 3J). These results suggest that the glycerophospholipid metabolism pathway mediated by LCAT may play a key role in maintaining sperm motility.

Fig. 3.

Alterations in SP metabolites between the high- and low-motility groups. Partial least-squares discriminant analysis (A), principal component analysis (PCA) (B), histogram (C), volcano plot (D), heat map (E), and KEGG enrichment scatter plot (F) showing the differentially abundant metabolites (DMs). KEGG and GSEA enrichment analysis of DMs (G–I). Content of lecithin-cholesterol acyltransferase (LCAT), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and free cholesterol (FC) in SP (J). Metabolites with log2(FC) ≥ 1.2 and p < 0.05 were considered significant DMs. Mean values with different letters differ significantly at p < 0.05 (Student’s t test, n = 6).

Analysis of Seminal Plasma Proteomics Results

The results of the Pearson correlation analysis between samples and the principal component analysis (PCA) of SP proteomics showed that the samples were well clustered (Fig. 4A). Based on log2FC ≥ 0.263 and p < 0.05, a total of 533 upregulated and 257 downregulated differentially expressed proteins were identified (Fig. 4B). The KEGG analysis of the differentially expressed proteins revealed that 113 pathways were enriched, of which 21 were significantly enriched. The upregulated differentially expressed proteins were enriched in 64 and 21 unique pathways in the SP of the HMG and LMG, respectively. According to the KEGG enriched pathways of differential proteins, the Top20 pathways with the lowest p-value were selected for the bubble diagram display (Fig. 4C). There were 21 items involved in the metabolic processes of HMG, mainly related to amino acid, carbohydrate, and phospholipid metabolism. 11 proteins were involved in cellular processes, mainly related to cell senescence and autophagy. 10 proteins were involved mainly in the mTOR, FoxO, MAPK, phosphatidylinositol, and Wnt signaling pathways. 15 metabolic processes were involved in LMG, mainly unsaturated fatty acid, carbohydrate, and glutathione metabolism. Ferroptosis was involved in cellular processes. The signaling molecules involved were extracellular matrix–receptor interactions, cell adhesion molecules, and cytokine-cytokine receptor interactions (Supplemental Table S3).

Fig. 4.

Combined seminal plasma proteomics and metabolomic analysis. Correlation analysis of sample protein expression levels (A), differential protein volcano plot (B), pathway bubble diagram (C), Venn diagram of KEGG enrichment pathways for proteomics and metabolomics (D), bubble map of protein metabolism co-enrichment pathways (E), Sankey diagram of glycerophospholipid metabolic pathways (F), detection of key substances in seminal plasma after co-culture with targeted activation of LCAT(G). Mean values with different letters differ significantly at p < 0.05 (Student’s t test, n = 6).

Joint Analysis of Seminal Plasma Proteome and Metabolic Pathways

Combined analysis of the seminal plasma proteome and metabolome revealed 30 co-enriched pathways (Fig. 4, D and E), of which 18 were metabolic pathways, mainly associated with lipid metabolism (glycerophospholipid metabolism and arachidonic acid metabolism) (Fig. 4F). The metabolites and proteins involved were phosphatidylserine/lysophosphatidylserine (PS/LPS), phosphatidylcholine/lysophosphatidylcholine (PC/LPC), phosphatidylethanolamine/lysophosphatidylethanolamine (PE/LPE), GPD1L2, LCAT, and L-PGDS. The metabolites and proteins involved in amino acid metabolism include tyrosine, arginine, γ-aminobutyric acid, glutamic acid, aspartic acid, GOT1, and CKB. There were 7 cell process pathways, primarily necroptosis and ferroptosis, and the metabolites and proteins involved were sphingomyelin, glutamate, CHMP4C, PPID, HMGB1, HSP90AA1, BID, and PRNP. Three environmental information pathways were identified, including the FoxO and mTOR signaling pathways. (Supplemental Table S4). The major metabolites in the glycerophospholipid metabolic pathway were PC/LPC, and the major protein was LCAT (Fig. 4F). Therefore, we detected the levels of these substances in the seminal plasma using an enzyme-linked adsorption assay (Fig. 4G). The LPC content was significantly higher in the LMG than the HMG, while PC and FC contents were significantly higher in the HMG than the LMG, verifying the reliability of the seminal plasma proteomic data.

LCAT Activation Impairs Sperm Function

Based on the alterations in the SP metabolites, the LCAT stimulator APOA-I was applied to explore the effect of LCAT on sperm function. Exposure to 0 to 0.01 mg/ml APOA-I at 37 °C caused no obvious difference in sperm motility; however, 0.10 to 1.00 ng/ml APOA-I decreased sperm motility (p < 0.05) compared to the NaCl group (Fig. 5A). Therefore, 0.10 mg/ml APOA-I was used for subsequent experiments. Incubation with NaCl for 2 h at 37 °C significantly reduced the content of PC in SP, which was further decreased by APOA-I (p < 0.05), along with a notable increase in LPC levels (Fig. 5, B and C). Transmission electron microscopy revealed that normal sperm heads presented a relatively uniform distribution of chromatin, intact plasma membranes, and normal mitochondrial morphology, whereas sperm incubated with APOA-I presented more ruptured plasma membranes, more chromatin loss, and swollen mitochondria (Fig. 5D). Compared to the NaCl group, APOA-I administration significantly increased the malformation rate of sperm and reduced membrane integrity (both p < 0.05; Fig. 5, E and F). In vivo, APOA-I treatment remarkably decreased both the longest fertilization days and the number of fertilized eggs in HMG, but not LMG (Fig. 5, G and H). These findings indicate that LCAT activation by APOA-I manipulation impaired the structure and function of sperm and compromised fertilization traits.

Fig. 5.

Modulation of lecithin-cholesterol acyltransferase (LCAT) activity through apolipoprotein A-I (APOA-I) supplementation on sperm characteristics. The effects of varying APOA-I concentrations on sperm motility were quantitatively assessed (A). The content of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in SP in response to 0.10 mg/ml APOA-I treatment (B, C). Transmission electron microscopy of sperm (D). Differences in the sperm malformation rate (E), membrane integrity (F), longest fertilization (G), and number of fertilized eggs (H) after co-incubation with APOA-I. Mean values with different letters differ significantly at p < 0.05 (Analysis of variance with Tukey’s post hoc test).

Discussion

Sperm motility is a major determinant of male fertility (2). It is one of the most important indicators for assessing sperm quality, fertilization, and semen preservation (3), and a major prerequisite parameter for sperm to navigate to the vagina and penetrate sperm storage tubules (41). SP is a complex biological fluid that has important effects on sperm function and survival (4). Some free proteins in SP bind the sperm membrane, which is crucial in SP function (42). Therefore, SP is considered a reliable indicator of male fertility. However, some studies have shown that seminal plasma negatively affects sperm preservation in vitro. Our findings in Yufen No. 1 roosters using both SP removal and exchange experiments demonstrated that SP plays an important role in sperm function and survival in vitro.

An intact sperm membrane is necessary for sperm motility (43, 44). Factors such as lipid peroxidation in the sperm plasma membrane can lead to reduced fertility (45). Lipid peroxidation products can significantly change the protein structure, leading to cellular metabolic disorders, dysfunction, and apoptosis (46). In avian spermiocytes, polyunsaturated fatty acids, such as arachidonic acid, are among the most abundant and are particularly sensitive to lipid peroxidation (47). Cholesterol is an essential lipid component that maintains the integrity, fluidity, and microstructure of cell membranes. Therefore, the regulation of lipid and cholesterol homeostasis in the cell membrane can achieve different cellular functions. SP free cholesterol (FC) content in HMG and LMG reveals dynamic changes in membrane lipids and cell function. Phospholipids, followed by sphingolipids and cholesterol, are major components of the cell membrane. The membrane lipid composition is highly diverse and dynamically regulated (48). Glycerophospholipids are important structural and functional components of cell membranes and are precursors of various lipid mediators (49). Several markers involved in cholesterol metabolism and transport were expressed to varying degrees in the studied tissue specimens, suggesting that cholesterol in SP may play an important role in spermatogenesis (50). Our results showed that SP cholesterol content significantly increased in both the LMG and after targeted activation of LCAT activity, suggesting that FC in SP may play an important role in the maintenance of sperm motility in vitro. Studies have shown that in the plasma, LCAT prevents the accumulation of unesterified cholesterol, and a significant increase in free cholesterol is accompanied by a significant decrease in esterified cholesterol (51). We observed high FC content and low LPC content in the SP of HMG, which verified that the role of LCAT in SP was the same as that in plasma. In addition, the incubation of intact sperm with SP activators showed significant changes in the content of SP and its metabolites in the semen, effectively regulating sperm motility and significantly affecting sperm membrane integrity. These results indicated that these key biomarkers regulate sperm motility by affecting sperm membrane homeostasis. However, a further explanation of the molecular mechanisms by which altered sperm membrane integrity alters sperm motility is required.

Lecithin cholesterol fatty acyl transferase (lecithin-cholesterolacyltransferase, LCAT) is released into the blood by the liver. LCAT is the only enzyme in plasma that can esterify cholesterol and promote its esterification with phosphatidylcholine-derived acyl chains. Its role is to transfer the C2 unsaturated fatty acid of lecithin from high-density lipoproteins (HDL) to free cholesterol, resulting in the formation of lysophosphatidylcholine and cholesteryl ester to maintain cholesterol homeostasis (52). LCAT can directly extract phospholipids from the surface of recombinant HDL in the same manner as phospholipase A2, which is the first substrate required for the cholesteryl ester exchange mechanism (53). LCAT is a soluble enzyme with phospholipase A2 activity (cleaving fatty acids at the sn-2 position of phosphatidylcholine and other phospholipids) and transester exchange activity (transferring cleaved fatty acids to the hydroxyl group on the A-ring of cholesterol). The most important physiological reaction is the conversion of cholesterol and phosphatidylcholine (PC) (lecithin) to cholesteryl ester and lysophosphatidylcholine (LPC) (54, 55, 56). Membrane lipids are primarily composed of cholesterol, phospholipids, and glycolipids, which play roles in cell adhesion and signal transduction at different stages of spermatogenesis (57). The total cholesterol content in human seminal plasma is positively correlated with sperm density, motility, and morphology (50). LCAT is expressed in small amounts in the brain and testis (58, 59) and its loss of function or mutation can cause various metabolic disorders. Mutations in LCAT are associated with familial LCAT deficiency (FLD) and fish eye disease (FED), and LCAT abnormalities can lead to dyslipidemia and arteriosclerosis (60). Since the mid-1990s, studies in transgenic animals overexpressing human LCAT have confirmed its importance in cholesterol homeostasis and suggested its potential as a therapeutic target for preventing or reversing atherosclerosis (61). However, the effect of LCAT on sperm motility in seminal plasma is unclear. We elucidated the changes in LCAT content in rooster SP and determined the conversion of PC to LPC in glycerophospholipid metabolism, establishing its role in the maintenance of sperm membrane structure and function and sperm motility in vitro.

APOA-I is the most potent activator of LCAT (62, 63). Co-incubation of sperm with APOA-I to target LCAT activity revealed an inverse correlation between sperm motility and APOA-I concentration. Higher concentrations were associated with a significant reduction in plasma membrane integrity and increased sperm malformation, indicating changes in the sperm membrane structure, which was further confirmed by transmission electron microscopy. To further confirm the regulation of sperm motility by the targeted activation of LCAT, we verified the effect of LCAT on the maintenance of sperm motility in hens using a paired hatching assay. Overall, the results suggest that LCAT activity accelerates the breakdown of sperm membrane phospholipids, which leads to the conversion of more PC to LPC and the consumption of a large amount of cholesterol, leading to changes in the membrane structure and function of sperm cells, further aggravating sperm apoptosis and decreasing motility. Therefore, we elucidated the role of LCAT in SP in the maintenance of sperm motility in vitro.

This study has two limitations. First, we attempted to use 4-hydroxynonenal (4-HNE) as an LCAT inhibitor to incubate sperm, and the results appeared promising. 4-HNE is a highly reactive α,β-unsaturated aldehyde formed during lipid peroxidation of PUFAs and is associated with reduced sperm motility (64). Because 4-HNE binding can inhibit LCAT, the observed effects on sperm motility may have resulted from LCAT inhibition or the oxidation of 4-HNE. Second, the role of amino acids and proteins in sperm properties remains unclear. SP metabolite analysis revealed the enrichment of some amino acid and protein pathways. Previous studies have shown that some proteins in SP are associated with semen quality and fertilization rates in male birds (5, 6, 7). However, our study did not provide functional data on the promotion or inhibition by certain proteins or amino acids. Therefore, some of our conclusions may overestimate or underestimate the contribution of critical pathways or LCAT in sperm motility. Further studies are needed to address these limitations.

Conclusion

We established, through proteomic and metabolomic analyses of SP, that LCAT is the key protein enriched in the glycerophospholipid pathway, and PC and LPC are the key metabolites. The SP protein LCAT transfers unsaturated fatty acids of sperm membrane phospholipids PC to cholesterol to form cholesteryl ester and LPC, changing cell membrane composition and function, affecting the structure of sperm membrane phospholipids, and regulating sperm motility. To the best of our knowledge, this study is the first to investigate the role of the SP protein LCAT in the maintenance of sperm motility in vitro, which serves as a great reference value for improving the formulation of poultry semen dilutions and improving the fertility of breeding roosters.

Data Availability

It is also available from the corresponding author upon reasonable request.

Supplemental data

This article contains supplemental data.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.