Abstract Background This study was designed to identify candidate marker proteins that influence the growth and development of Shandong black cattle bull testes through multiomics joint analysis, thereby providing a certain theoretical basis for testis growth and development as well as bull selection. Eight 12-month-old Shandong Black cattle bulls were selected, and testis tissues were collected. The testes were categorized into two groups on the basis of their morphological characteristics: Group 1 (weight > 120 g) and Group 2 (weight < 120 g), with 4 animals in each group. Group 2 was employed as the control group to construct a protein and metabolite library for joint analysis to screen candidate marker proteins that affect testis spermatogenesis. Results The results revealed that 1553 differential expression proteins (DEPs) were differentially expressed between the large and small testes of black Bleykett bulls, with 1219 being upregulated and 334 being downregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results revealed that the upregulated DEPs were involved primarily in the cell cycle (CDK1, CCNB, MCM4), DNA replication (MCM3, MCM4), etc. The downregulated DEPs were associated mainly with metabolic pathways (ACSM1, IMPDH1), etc. The Gene Ontology (GO) enrichment results revealed that the DEPs were significantly enriched in the categories of cytoskeleton movement. Weighted gene coexpression analysis suggested that testis weight was significantly correlated with MCM, STRADA, and SEC31B. After the DEPs were integrated, a protein–protein interaction (PPI) analysis was performed, and 10 key regulatory proteins, including MCM3, MCM4, CDK1, and CDK2, were identified. Metabolomics demonstrated that 14 upregulated metabolites were predominantly enriched Glycerolipid metabolism (uridine diphosphate glucose), and 59 downregulated metabolites were significantly enriched in metabolic pathways (hypoxanthine). Conclusion A combined analysis revealed that UDPG upregulation enhances MCM3/MCM4 activity during S phase, thereby promoting spermatogenesis. Hypoxanthine upregulation inhibits the activity of CDK1, leading to a blockage in the transition from the G2/M phase of the cell cycle, thereby inhibiting spermatogenesis. In summary, MCM3, MCM4, and CDK1 participate in regulating the process of testis spermatogenesis. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-025-11825-1. Keywords: Multiomics, Shandong black cattle, Testis, Spermatogenesis Introduction The testes constitute the core organ of the male reproductive system, exerting exocrine functions in terms of sperm production and endocrine functions as part of the hypothalamus‒pituitary‒gonad axis via testosterone production [[42]1, [43]2]. The phenotypic indicators of the testes include testis weight, testis length, and testis width, among which testis weight is one of the most critical indicators for evaluating the reproductive ability of bulls [[44]3]. In production practice, testis weight is frequently utilized as an important trait for exploring the development of the testes. The increase in testis weight is directly related to the development of the seminiferous tubule epithelium, which offers a significant site for sperm production [[45]2, [46]3]. Therefore, the testis weight can affect the sperm quantity and quality, and it is an important phenotypic trait for judging whether the testicular development is affected in animal experiments. In conclusion, exploring candidate marker proteins that affect testis weight has a significant role in enhancing the reproductive ability of bulls and facilitating scientific breeding selection. In recent years, the number of studies on the diverse genomics of the testis has increased continuously. Research has demonstrated that the EPHB6 (Ephrin Type-B Receptor 6) and TLR1 (Toll-Like Receptor 1) genes in the distal epididymis of the bull have the potential to regulate the stability of testicular cells and sperm maturation. Through the exploration of genes such as EPHB6 and TLR1, the heritability of sperm quality in bulls can be improved, and their reproductive ability can be augmented [[47]4]. Xiong et al. [[48]5] employed DIA (data-independent acquisition) proteomics to identify differentially expressed proteins in PRSS37-deficient testes and sperm, revealing predominant associations with energy metabolism pathways, thereby highlighting PRSS37-mediated metabolic regulation in male fertility. Zheng et al. [[49]6] applied iTRAQ proteomics to testis-specific knockout flies of ocnus, revealing altered testicular proteomes mechanistically linked to mitochondrial homeostasis, Notch signaling, and apoptosis/autophagy pathways, thereby offering molecular insights into male testis development. Research has revealed that the TM4 Sertoli cells metabolome of the testis is compromised by PM2.5 due to metabolic disorders and oxidative stress, leading to the inhibition of TM4 Sertoli cells proliferation and functional deterioration, directly impairing male reproductive ability [[50]7]. Wang et al. [[51]8] revealed through the analysis of the sheep serum metabolome and the small intestine-testis axis that rumen microbial activity is positively correlated with the sperm motility of sheep. The sperm quality and motility of ram can be regulated by influencing rumen L-tryptophan metabolism, thereby increasing ram fertility. Transcription factors, proteins, and metabolic products can influence testicular development, and different genomics methods provide technical support for the study of testicular development. Moreover, the underlying molecular mechanisms have been clearly elucidated. Nevertheless, in recent years, the majority of studies on the testes have relied on single-omics analysis, and relatively few studies have explored the association between testis weight and spermogenesis in the Shandong black cattle. Hence, this study aimed to explore the candidate marker proteins influencing testis spermogenesis in Shandong black cattle bulls from a multiomics perspective, which could provide a certain theoretical foundation for the selection of Shandong black cattle. In this study, the Shandong black cattle utilized were an elite beef cattle population bred via modern biological technology [[52]9]. This study implemented a multiomics joint analysis by performing phenotype observations, screening differential expression proteins (DEPs), annotating the GO (Gene Ontology) functions of differentially expressed proteins, and enriching Kyoto Encyclopedia of Genes and Genomes (KEGG) signalling pathways, with the aim of selecting Differentially expressed proteins and metabolites that have an impact on bull testis spermatogenesis. Additionally, the candidate proteins influencing the testis phenotype were further screened through the use of bioinformatics methods such as Weighted gene coexpression network analysis (WGCNA) and protein–protein interaction (PPI). This study organically combines multiple omics technologies, conducting analyses and screening of candidate proteins that influence various aspects of sperm development. This approach will be more conducive to the exploration and selection of relevant markers, as well as the enhancement of animal traits and scientific breeding and selection. Materials and methods Experimental animals The experimental animals were 8- to12-month-old bulls selected from Shandong Zhaofu Animal Husbandry Technology Co., Ltd. in Shandong Province, China. The cattle shed had good lighting and ventilation, and the temperature and humidity were suitable. The feeding conditions were kept consistent. All animals were handled in accordance with ethical guidelines to ensure their welfare throughout the study. The current study was approved by Committee on the Ethics of Animal Experiments of Qingdao Agricultural University IACUC (protocol code DKY20220805 and date of 2022.08.05). Testicular samples were collected, and their weight, long diameter and wide diameter were measured. The testicular samples were stored in liquid nitrogen and paraformaldehyde. The bulls were divided into 2 groups according to the critical value of 120 g for testicular weight, with 4 bulls in each group. Group G1 was the large testicle group, and Group G2 was the small testicle group (control group). Tissue observation of testis samples Testis samples preserved in paraformaldehyde were fixed in Bouin's solution for 12 h. The fixed tissue was dehydrated with graded alcohol, cleared with xylene, embedded in paraffin, cut into sections, stained with hematoxylin and eosin dyes(HE) [[53]10], and finally sealed and observed under a microscope, and photos were taken. Proteomic analysis of testes Testicular sample preparation was conducted via the iST Sample Preparation Kit (PreOmics, Germany). Quality control of the raw mass spectrometry data was performed via QuiC software (Biognosys) [[54]11]. A library of data obtained from the DDA (PHnano-HPLC-MS/MS) acquisition mode (The mass spectrometry parameters are set as follows: (1) MS: Scan range (m/z): 350—1500; Resolution: 120,000; AGC target: 4e5; Maximum injection time: 50 ms; Dynamic exclusion time: 30 s; (2) HCD-MS/MS: Resolution: 15,000; AGC target = 5e4; Maximum injection time: 35 ms; Collision energy: 32.) was constructed via Pulsar software [[55]12], with a Q value (FDR) threshold of 1% for both the precursor and protein levels. DIA data (The mass spectrometry parameters are set as follows: (1) MS: Scan range (m/z): 350—1500; Resolution: 120,000; AGC target: 4e6; Maximum injection time: 50 ms; (2) HCD-MS/MS: Resolution: 30,000; AGC target: 1e6; Collision energy: 32; Energy increase: 5%. (3) Variable window acquisition, 60 windows are set, overlapping serial ports are configured, and each window overlaps by 1 m/z.) were analysed against the DDA reference database to identify proteins, applying a Q value cut-off of 1% at both the precursor and protein levels as the identification criterion. T-test was used for initial significance assessment of proteomic differential expression analysis, followed by multiple hypothesis tests calibrated by Benjamini-Hochberg method. Only the results of |Fold Change|≥ 1.5 and Qvalue (FDR) ≤ 0.05 were retained. A Q value ≤ 0.05 was set as the threshold for identifying DEPs. GO functional annotation analysis and KEGG signalling pathway enrichment analysis were conducted via the R package clusterProfiler [[56]13]. The results were visualized via the ggplot2 package [[57]14] in R. Metabolomic analysis of testes Testicular tissue was subjected to metabolite extraction and analysed via an LC‒MS system. The raw data were processed through peak identification, peak filtering, and peak alignment. Background ions were removed from the blank samples, and the quantitative results were normalized. The final identification and quantification of metabolites were performed. Quality control (QC) samples were used throughout the testing process to ensure data reliability (RSD ≤ 30%, PCA clusters well). On the basis of high-resolution mass spectrometry (HRMS) detection technology, metabolites were identified by matching molecular characteristic peaks against high-quality databases, including mzCloud, mzVault, and MassList, constructed from standard substances. Qualitative and quantitative analyses of the metabolites were then performed. Differential expression analysis of metabolites was conducted via the R package ropls [[58]15]. Only the results of VIP ≥ 1, |Fold Change|≥ 1.5 and Qvalue (FDR) after correction by Benjamini-Hochberg ≤ 0.05 were retained. Principal component analysis (PCA) of G1 and G2 was performed via the R package gmodels (v2.18.1) [[59]16]. For the identified differentially abundant metabolites, a Q value ≤ 0.05 was set as the threshold, and KEGG signalling pathway enrichment analysis was conducted via the R package clusterProfiler [[60]13]. The results were visualized via the ggplot2 package [[61]14] in R. WGCNA The WGCNA method [[62]17] in the R package was used to construct a coexpression network for the total DEPs of the proteome, with the following parameters: corType ="pearson", minModuleSize = 30, and mergeCutHeight = 0.1. After the genes were filtered, hierarchical clustering analysis was performed via the R package gplots [[63]18]. A standard scale-free network was established with a soft threshold of power = 8. Correlation analysis was conducted between module characteristic genes and testicular phenotypic data. Modules with Pearson correlation coefficients closest to 1 (correlation test p < 0.05) were selected as representative modules on the basis of this criterion. PPI network enrichment analysis The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database [[64]19] was used to construct a PPI network from the screened DEPs. Cytoscape software was used for visual analysis, and its plugins CytoHubba and CytoNCA were utilized to identify the highest-degree hub proteins in the network. Joint analysis was conducted between the key hub proteins identified via Cytoscape and the key module proteins identified via WGCNA. Immunohistochemical Assay Testicular tissues (approximately 1 cm^3) were embedded in paraffin and sectioned. Paraffin sections were dewaxed with eco-friendly dewaxing agents (three times, 10 min each), dehydrated through graded ethanol (three times, 5 min each), and rehydrated. After antigen retrieval and natural cooling, sections were washed in PBS (pH7.4) with shaking (three times, 5 min each). Endogenous peroxidase activity was blocked by incubation in 3% H[2]O[2](25 min, dark), followed by PBS washes and blocking with 3% BSA (30 min, RT). Primary antibody incubation was performed at 4 °C overnight in a humidified chamber, followed by HRP-conjugated secondary antibody incubation (50 min, RT). DAB staining was microscopically monitored and terminated by rinsing under running water. Nuclei were counterstained with hematoxylin (3 min), differentiated, and blued. Sections were dehydrated, cleared, mounted, and analyzed under a light microscope [[65]20]. The average optical density value was detected by Image-J image analysis software. Western Blot analysis A protein extraction kit was used to extract protein samples from various tissues and testes. Twenty micrograms (20 µg) of protein samples were loaded onto a 12% SDS‒PAGE gel for electrophoresis and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 3 h at room temperature. The samples were then incubated overnight at 4 °C with primary antibodies against β-actin, CDK1 (Cyclin-dependent kinases 1), CDK2 (Cyclin-dependent kinases 2), MCM3 (Microchromosome maintenance protein3), MCM4 (Microchromosome maintenance protein4), ACOT7 (Acyl-CoA Thioesterase 7), and RAP2C (Ras-Related Protein Rap-2c). After antibody incubation, the PVDF membranes were washed three times with TBST and then incubated with secondary antibodies in the dark at room temperature for 2 h. The membranes were subsequently washed again three times with TBST and once with distilled water. Chemiluminescence and development were performed using a luminescent solution. The gray values of the target bands were analysed via ImageJ software. Real-time fluorescence quantitative PCR (qPCR) RNA extracted from various tissues was reverse transcribed and subjected to qPCR. The qPCR system (20 µL) consisted of 10 µL of 2 × SYBR Green Pro Taq HS Premix II, 0.4 µL each of forward and reverse primers (10 µmol/L), 1 µL of cDNA, and 8.2 µL of ddH2O. The thermal cycling conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing and extension at 60 °C for 30 s. The internal reference gene GAPDH was amplified under the same reaction conditions. The relative expression levels were calculated via the 2^(-ΔΔCt) method. Statistical analysis The data were analysed via R software. The results are expressed as the means ± SEMs and include at least three independent biological replicates (n = 3), and the data were analysed via independent sample t tests. P < 0.05 was considered the criterion for significance. Trial data plotting was performed with GraphPad Prime 10, etc. Results Statistical analysis of phenotypic data On the day of castration for 12-month-old bulls, measurements were taken to assess the weight, width, and length of the testicular samples. Body weight was recorded on the day of castration (0 d). Independent sample t tests revealed significant differences (P < 0.05) between the two groups in terms of body weight, testis weight, testicular index (testicular weight/body weight × 100%), testis width, and testis length (Fig. [66]1d). The most significant discrepancy occurred in testis weight, whereby the G1 group exhibited a testis weight 1.8 times that of the G2 group. It was also found that the testicular index of G1 group was twice that of G2 group. In contrast, differences in testis width and length were relatively small, with each measurement in the G1 group being approximately 1.2 times greater than that in the G2 group. Notably, the G2 group had a body weight that was 1.1 times greater than that of the G1 group at 0 d. Fig. 1. [67]Fig. 1 [68]Open in a new tab Phenotypic analysis. a Summary of test process. b Hematoxylin and eosin dyes staining images of G1 testicular tissue. c Hematoxylin and eosin dyes staining images of G2 testicular tissue. d Columnar chart of testicular phenotypic data. e Columnar chart of SC count. f Columnar chart of SP count. g Columnar chart of IC count. h Columnar chart of RS count. i Columnar chart of PS count. For (b-c), In group G1, the lumen of the seminiferous tubules of the testis was filled and the cells were closely arranged. In group G2, the vacuolation of the lumen of the seminiferous tubules of the testis increased and the cells were arranged sparsely and chaotically. The cell density in group G1 was significantly higher than that in group G2. For (d-e), Testicular weight and Testicular index were unilateral testis. For (d-k), Asterisk (*) means significant difference (P < 0.05), (**) means significant difference (P < 0.01), (***) means significant difference (P < 0.001).ns means no significant difference. For (b-c, e-i), Hematoxylin and eosin dyes staining images are shown as testicular seminiferous tubule, SC: Sertoli cells, LC:Leydig cells, SP: spermatogonium, PS: primary spermatocyte, RS: round spermatids, S: Mature sperm Testicular tissue staining HE staining revealed that the lumens of the seminiferous tubules in the G1 group were fully occupied, and the bases of the Sertoli cells near the basal membrane were orderly arranged around the spermatogenic cells, with the spermatogenic cells closely arranged. Spermatogonial cells were positioned near the basal membrane, round spermatids were abundant, and Leydig cells were abundant and swarmed. (Fig. [69]1b). In contrast, the seminiferous tubules in the G2 group presented enlarged luminal spaces, sparse and disorganized Sertoli cells and spermatogenic cells, a reduced number of round spermatids, and sparse Leydig cells. (Fig. [70]1c). The numbers of LCs, SCs, SPs, PSs and RSs in the testes of the G1 group were significantly greater than those in the testes of the G2 group (Fig. [71]1f-j). In the G1 group, the number of testicular SCs was 1.9 times greater than that in the G2 group, the number of RSs was 2 times greater than that in the G2 group, the number of LCs and SPs was approximately 1.8 times greater than that in the G2 group, and the number of PSs was approximately 3 times greater than that in the G2 group. Differential protein analysis of the testicular proteome DEPs were identified on the basis of the criteria of |Fold Change|≥ 1.5 and p < 0.05 from the proteomic data. The heatmap (Fig. [72]2a) demonstrated excellent sample reproducibility and significant differences between groups, facilitating subsequent data analysis. A volcano plot (Fig. [73]2b) was constructed on the basis of the expression levels of the DEPs. In the G1 group, 1219 proteins, including members of the CDK (Cyclin-dependent kinases) family (e.g., CDK1, CDK2), CCNB2 (Cyclin B2), CCNB1 (Cyclin B1), and the MCM (Microchromosome maintenance protein) family, were upregulated, whereas 334 proteins, including ALDH18A1 (Aldehyde Dehydrogenase 18 Family Member A1), were downregulated. Fig. 2. [74]Fig. 2 [75]Open in a new tab Differential protein analysis of testicular proteome. a Differential protein heat map; (b) Differential protein volcano map; (c) Enrichment analysis of differentially up-regulated protein KEGG; (d) Enrichment analysis of differentially down-regulated protein KEGG; (e) Differentially up-regulated protein GO analysis; (f) Differentially down-regulated protein GO analysis; (g). Module division; (h) Correlation analysis of module. (i). Differential protein PPI analysis, PPI analysis of protein and WGCNA representative modular protein Venn diagram. For (a),Heat map colors represent log2 normalized values for gene expression (see plot note on the right). Red indicates high expression, blue indicates low expression, and values range from-2 (lowest) to + 2 (highest). For (b), a yellow and blue dot indicates that the p-value and log2(FC) of the DEPs reached the threshold,Gray dots indicate insignificant differences. For (c), The left side of the Sangkey diagram indicates the KEGG pathway name. The right X-axis represents the Rich factor, with the size of the points corresponding to the count. The Y-axis represents pathway significance. In the GO enrichment analysis diagram(d), the X-axis represents the name of the protein enrichment GO class. The y axis represents the protein count. Red represents biological processes, green represents cell classification, and blue represents molecular function. For (e–f), Co-expression modules related to testicular phenotype were constructed and identified based on 1553 DEPs. For (g), Different colors of the WGCNA cluster tree represent different modules. The correlation between modules and testicular phenotypes is represented by different colors and numbers (h). Pearson correlation coefficient method was used to estimate the correlation. For (i) PPI analysis of 240 DEPs. Near the center of the PPI circle is hubgene; On Venn diagram each circle shows the differential proteins contained in the representative module or PPI, and the overlap of the circles is the common differential proteins Functional analysis of DEPs KEGG pathway enrichment analysis and GO enrichment analysis were performed on the DEPs. The KEGG enrichment results indicated that the upregulated DEPs in the G1 group were involved primarily in pathways such as the cell cycle (e.g., CDK1, CCNB1, MCM3, and BUB1B), DNA replication (e.g., MCM family), p53 signalling pathway (e.g., CDK1, CDK2, and CCNB1), and others (Fig. [76]2c). The downregulated DEPs in the G1 group were associated mainly with metabolic pathways (e.g., ACSM1 and IMPDH1), arginine and proline metabolism (e.g., ALDH18A1 and CKB) and other pathways (Fig. [77]2c). The GO enrichment results revealed that DEPs were significantly enriched in 45 GO terms, including “cellular processes” (e.g., STRADA, SEC31B), “cellular anatomical entities” (e.g., MAP2K5, IMPDH1), and “cytoskeletal motor activity” (e.g., kIF11, MYO6) (Fig. [78]2d). After the significant KEGG pathways and GO terms were integrated, a total of 240 DEPs were selected for further analysis. Screening of representative modules of DEPs via WGCNA The 1553 DEPs identified on the basis of the criteria of |Fold Change|≥ 1.5 and p < 0.05 were divided into five gene modules and subjected to correlation analysis (Fig. [79]2g). By correlating each module with testis weight, testis width, and testis length, representative modules were selected. The results indicated that testis weight was significantly correlated with the representative modules"MEturquoise"(r = 0.74, p = 0.02) and"MEbrown"(r = −0.82, p = 0.004) (Fig. [80]2h). Specifically, the"MEturquoise"module contained 1059 DEPs, including MCM4, MCM3, and CDK1, whereas the"MEbrown"module included 95 DEPs, such as SDF2L1, PDCL2, and HYOU1. Results of the PPI analysis of the DEPs After the significant KEGG enrichment pathways and GO enrichment terms from the proteomic data were integrated, 240 DEPs were selected. The STRING online tool was used for PPI network analysis of these DEPs, and the results are shown in Fig. [81]2i. Proteins such as MCM3, MCM4, MCM5, MCM6, CDK1, CDK2, CCNB1, CCNB2, CDK7, CCNH (Cell cyclin H), BUB1B (BUB1-Related Kinase 1), GTF2B (General Transcription Factor 2B), GTF2H5 (General Transcription Factor 2H), and RAN form the core of the regulatory network. Integrating PPI analysis with the representative modules from WGCNA revealed that 156 DEPs in the"MEturquoise"module had a significant positive correlation with testis weight, whereas 18 DEPs in the"MEbrown"module had a significant negative correlation with testis weight (Fig. [82]2i). Notably, all key regulatory proteins at the core of the network were found within the"MEturquoise"module, which was significantly positively correlated with testis weight. The main functions of these key regulatory proteins are summarized in Table [83]1 on the basis of relevant research reports. Table 1. Key proteins identified via combined PPI and WGCNA Network regulatory proteins GO Term KEGG Pathway Main function of protein CDK1/2/7 Biological Process: cellular process/developmental process/ Molecular Function:binding Cellular Component:protein-containing complex/cellular anatomical entity Cell cycle, P53 signaling pathway Cyclin-dependent protein kinase (CDK) is an important regulator of transcription, metabolism and cell differentiation in higher eukaryotic cells [[84]21]. CDK 1 is a potential dynamin 2 (DNM2) phosphorylated kinase in mammalian testis, which affects the meiosis of testis germ cells [[85]22] CCNB1/2 Biological Process: cellular process Molecular Function: molecular function regulator Cellular Component: cellular anatomical entity Cell cycle In yeast and animals, cyclin B binds to and activates cyclin dependent kinase (CDK) to drive entry into mitosis [[86]23]. CCNB1 and CCNB2 are involved in all stages of spermatogonial mitosis [[87]24] and are necessary for germ cell and spermatogonial proliferation [[88]25] BUB1B Biological Process: cellular process/metabolic process Molecular Function: binding/catalytic activity BUB1B, which is localized to the centromeres, is a component of the mitotic checkpoint complex (MCC) and an inhibitor of the late promoting complex/ring (APC/C) to control mitosis [[89]26]. Increased expression of BUB1B/BUBR1 contributes to abnormal DNA repair activity, thereby generating resistance to DNA damage factors [[90]26] CCNH Biological Process: cellular process/regulation of biological process Molecular Function: general transcription initiation factor activity Cellular Component: protein-containing complex Cell cycle, Basal transcription factors Cell cyclin H (CCNH) may exist as a stable protein in immature oocytes, which is necessary for oocyte maturation [[91]27]. The complex formed by cyclin H and CDK7 exists in testicular cells and participates in the meiosis process [[92]28] MCM2-6 Biological Process: reproduction /reproductive process/developmental process Molecular Function: binding/catalytic activity Cellular Component: protein-containing complex/cellular anatomical entity Cell cycle, DNA replication Microchromosome maintenance protein (MCM) is a DNA-dependent ATPase that binds to the replication origin and supports DNA replication. A large amount of MCM2-7 is assembled on chromatin in G1 phase into a pre-replication complex called a"replication license"[[93]29], part of which becomes a helicase complex required for genome replication to promote sister chromatid cohesion in S phase [[94]30] GTF2B/GTF2H5/GTF2H2/GTF2F2/GTF2F1 Biological Process: cellular process/viral process Molecular Function: binding/catalytic activity Cellular Component: protein-containing complex/cellular anatomical entity Basal transcription factor GTF is a general transcription factor subunit of RNA polymerase II. It plays an important role in transcriptional initiation and nucleotide excision repair (NER) [[95]31] TAF4/9 Biological Process: reproduction/reproductive process/cellular process Molecular Function: ATP-dependent activity/binding Cellular Component: protein-containing complex/cellular anatomical entity Testicle-specific TPP-associated factors (tTAFs) regulate the transcription of genes necessary for spermatocyte entry into meiosis [[96]32]. TAF9 is involved in the histone acetylation complex and plays an important role in the completion of spermatogenesis [[97]33] RAN Biological Process: cellular process/localization/metabolic process Molecular Function: binding/catalytic activity/molecular function regulator Cellular Component: cellular anatomical entity Nucleoplasmic transport RAN (RAS-Associated Nucleoprotein) is a small GTP-binding protein belonging to the Ras superfamily that is critical for translocation of RNA and proteins through nuclear pore complexes [[98]34]. RAN proteins are also involved in controlling DNA synthesis and cell cycle progression, and have become central regulators of mitosis and meiosis [[99]34] [100]Open in a new tab Differentially abundant metabolite analysis of the testicular metabolome Differentially abundant metabolites were identified on the basis of the criteria of VIP ≥ 1, |Fold Change|≥ 1.5, and p < 0.05 in the metabolomic analysis. Principal component analysis (PCA) revealed significant differences in metabolite profiles between the G1 and G2 groups (Fig. [101]3a), facilitating subsequent data analysis. Using G2 as the control group, further analysis revealed that in the G1 group, there were 14 upregulated metabolites and 59 downregulated metabolites (Fig. [102]3b). The significantly upregulated metabolites in the G1 group included testosterone (fold change of 7), uridine diphosphate glucose (fold change of 2), etc. The significantly downregulated metabolites in the G1 group primarily consisted of creatinine, L-lysine, L-tyrosine, L-phenylalanine, hypoxanthine, nicotinamide, etc., with a fold change of approximately 2. The metabolite with the smallest difference was citric acid, with a fold change of 0.66. Fig. 3. [103]Fig. 3 [104]Open in a new tab Differential metabolites analysis of testicular metabolome. a metabolome principal component analysis; (b) volcanic map of differential metabolites. c KEGG up-regulation and down-regulation analysis of differential metabolites. For (b), a yellow and green dot indicates that the p-value and log2(FC) of the DEPs reached the threshold, black dots indicate insignificant differences. For (c), The left side of the Sangkey diagram indicates the KEGG pathway name. The right X-axis represents the Rich factor, with the size of the points corresponding to the count. The Y-axis represents pathway significance Functional analysis of differentially abundant metabolites KEGG pathway enrichment analysis was performed on the differentially abundant metabolites (Fig. [105]4c), with the upregulated differentially abundant metabolites enriched mainly in the androgen and estrogen receptor agonist/antagonist pathway (testosterone), the Glycerolipid metabolism (uridine diphosphate glucose), etc. The downregulated metabolites enriched mainly in pathways related to the biosynthesis of phenylalanine, tyrosine, and tryptophan (indole, L-tyrosine, L-phenylalanine), the 2-hydroxyacid metabolism pathway (L-methionine, L-tyrosine), and the metabolic pathway (hypoxanthine). Fig. 4. [106]Fig. 4 [107]Open in a new tab Western Blot and qPCR verification. a Immunohistochemical localization of CDK1, MCM3/MCM4 in testicular tissue. b Comparison of the trend of differential protein DIA and Western Blot. c Western Blot verified the expression levels of ACOT7, RAP2C, CDK2 and β-actin in large and small testis. d Analysis of CDK1 protein expression. e Analysis of MCM3 protein expression. f Analysis of MCM4 protein expression. g Western Blot verified CDK1 and MCM3 Expression of MCM4 in various tissues. h Validation of CDK1 transcription levels. i Validation of MCM3 transcription levels. j Validation of MCM4 transcription levels. For (a), SC: Sertoli cells, LC:Leydig cells, SP: spermatogonium, PS: primary spermatocyte, RS: round spermatids, S: Mature sperm. For (a-i), Asterisk (*) means significant difference (P < 0.05), ns means no significant difference Immunohistochemical results Based on the immunohistochemical results (Fig. [108]4a), it can be known that the positive expression intensities of CDK1, MCM3, and MCM4 in the testes of the G1 group were significantly higher than those in the G2 group (P < 0.05). In the G1 group, CDK1 was mainly localized in the cytoplasm and nucleus of spermatogonia, primary spermatocytes, and secondary spermatocytes. In the G2 group, CDK1 exhibited weak positive expression in the cytoplasm of Leydig cells, various spermatogenic cells, and Sertoli cells. MCM3/MCM4 were expressed in the cytoplasm and nucleus of Leydig cells, various spermatogenic cells, and Sertoli cells, and presented strong positive expression in Leydig cells, Sertoli cells, and spermatogonia. Western Blot and qPCR verification results To validate the proteomic data, CDK2, RAP2C, and ACOT7 proteins were selected for Western Blot verification based on comprehensive bioinformatics analysis. As shown in Fig. [109]4b, the Western Blot results for these three proteins were consistent with the trends observed in the proteomic data, confirming the reliability of the proteomic findings. The candidate proteins CDK1, MCM3, and MCM4 were further verified by Western Blot across various tissues. In the G1 group, MCM3 expression was highest in large testes and heart, while it was lowest in muscle (Fig. [110]4e). CDK1 exhibited high expression in lungs and kidneys, with the lowest expression in muscle (Fig. [111]4d). MCM4 showed the highest expression in large testes of the G1 group, followed by heart and lungs, with the lowest expression in muscle (Fig. [112]4f). qPCR verification results indicated that both CDK1 and MCM4 genes had the highest expression levels in testicular tissue, with significantly higher expression in the G1 group compared to the G2 group (Fig. [113]4g, i). The expression level of MCM3 was highest in the spleen, followed by testis in both G1 and G2 groups (Fig. [114]5h). Notably, all three genes were significantly underexpressed in heart, lliver and muscle. Fig. 5. [115]Fig. 5 [116]Open in a new tab MCM3, MCM4 and CDK1 regulate spermatogenesis Discussion The weight of the testes serves as a critical indicator for evaluating testicular growth and development, sperm quality and quantity, and reproductive performance in bulls [[117]2]. Furthermore, sperm quality and quantity directly influence reproductive performance. This study revealed that the most pronounced difference in testis weight was observed between the G1 and G2 groups. Additionally, bulls in the G2 group exhibited a more stable disposition, greater feed intake, and significantly greater body weight than those in the G1 group did. HE staining revealed that the spermatogenic tubules in the G1 group were more fully occupied, with closely arranged spermatogenic cells and a better-developed spermatogenic system. Franca et al. [[118]35] reported that testis weight in pigs was positively correlated with the area of the spermatogenic tubules, the thickness of the spermatogenic epithelium, and the number of reproductive cells. Greater testis weight is associated with a more developed vascular system, which facilitates sperm maturation [[119]36, [120]37]. In conclusion, testis size reflects the development of spermatogenic tubules and the vascular system, thereby influencing sperm production and providing a valuable reference for selecting Shandong black cattle for production. This study identified 1553 DEPs between the large and small testis tissues of Shandong black cattle, with 1219 upregulated and 334 downregulated. The Western Blot results confirmed the reliability of the proteomic data. Among the upregulated proteins, 154 DEPs were associated with reproductive processes, such as the CBS (Cystathionine Beta-Synthase) protein, which stimulates the proliferation, differentiation, and migration of endometrial epithelial cells and stromal cells by regulating endogenous hydrogen sulfide levels in yak uterine tissue, thereby maintaining the stability of the reproductive cycle in female mammals [[121]38]. Additionally, 106 DEPs related to spermogenesis, including KIF11 (Kinesin Family Member 11), which regulates spindle formation through microtubule acetylation during mitosis, impact the sperm production process [[122]39]. The remaining upregulated DEPs were involved in immune system regulation, antioxidation, catalytic activity, and other functions. Among the downregulated proteins, 180 DEPs were linked to metabolic processes, such as ALDH6A1 (Aldehyde Dehydrogenase 6 Family Member A1), which influences mitochondrial respiratory chain reactions in tumor cells, modulating the oxidation‒reduction balance [[123]40]. 31 DEPs were associated with biological adhesion, including CDH (Cadherins), which promotes the migration and aggregation of primordial germ cells and pre-Sertoli cells, affecting gonadal rudiment development and pre-Sertoli cells formation [[124]41]. The remaining downregulated DEPs were related to stimulus response, structural molecular composition, and other functions. In conclusion, the upregulated DEPs in reproductive processes and spermogenesis promote testis tissue growth, development, and sperm production, closely correlating with the testis phenotype. Conversely, the downregulated DEPs may disrupt metabolic processes, cell adhesion, and other functions, thereby influencing the normal growth and development of the testis. This study conducted a comprehensive analysis of the DEPs through GO and KEGG enrichment analyses. The GO enrichment results indicated that DEPs were significantly enriched in categories such as cytoskeletal motor activity. Actin generates mechanical force to shape the acrosome and sperm nucleus, maintain the movement of reproductive cells, and Sertoli cell connections. It also forms unique axial microtubule structures in the sperm tail to facilitate sperm motility in diverse fertilization environments [[125]42]. Cytoskeletal motor activity not only supports the morphological structure of reproductive cells but also contributes to sperm development. The KEGG enrichment results demonstrated that the upregulated DEPs were prominently enriched in pathways related to cell cycle regulation, including the cell cycle (e.g., MCM4, CDK1, CCNB1, and CCNB2), DNA replication (e.g., MCM3 and MCM4), p53 signalling pathway (e.g., CDK1, CDK2, and CCNB1) and others. Chen et al. [[126]43] reported that CCNB inhibits cell cycle progression under hypoxic conditions, affecting spermatogenic Leydig cell proliferation. Inhibiting the expression of the cell cycle regulatory factors CCNB1 and CDK1 can arrest cell cycle progression at the G2 phase, inhibit Sertoli cell proliferation, and induce apoptosis [[127]44]. These findings imply that CCNB and CDK1 facilitate the G2/M phase transition of the cell cycle, promote androgen production by Leydig cells, maintain the blood‒testis barrier, support the connection between Sertoli cells and germ cells, and promote spermatogenesis in the testis. MCM3 and MCM4 play crucial roles in DNA replication during S phase of the cell cycle. MCM is commonly used as a marker of cell proliferation in research [[128]45], and studies have shown significant expression of MCM transcripts in the testis [[129]46], influencing the growth and proliferation of Sertoli cells and Leydig cells [[130]43, [131]44]. These findings suggest that MCM3 and MCM4 drive Sertoli cell and Leydig cell proliferation via S-phase regulation, thereby facilitating spermatogenesis. Studies have shown that p53 responds to various stresses by inducing apoptosis, DNA repair and cell cycle arrest, and phosphorylation activation of P53 can inhibit CCNB1 activity and affect G2/M phase transition of cell cycle [[132]47]. CDK1/CCNB1 activity is negatively correlated with the phosphorylation level of p35, which regulates the cell cycle process [[133]48]. These results indicated that CDK1/CCNB1 affected the cell cycle process by P53 signaling pathway, and affected the growth and proliferation of sertoli cells and Leydig cells, and then affected testicular spermatogenesis. A pronounced downregulation of DEPs was observed in metabolic pathways, such as those involving ACSM1(Acyl-CoA Synthetase Medium-Chain Family Member 1) and IMPDH1(Inosine Monophosphate Dehydrogenase 1). ACSM1 has been verified to be specifically and significantly upregulated in prostate cancer, where it uses fatty acid oxidation to generate energy for cancer cell proliferation [[134]49, [135]50]. Moreover, normal germ cell metabolism typically employs glycolysis and the tricarboxylic acid (TCA) cycle to sustain the normal cell cycle [[136]51, [137]52]. Increased ACSM1 expression may disrupt germ cell metabolism, leading to abnormal cell cycles in testicular Leydig cells and Sertoli cells, thereby affecting normal spermatogenesis in the testis. The molecular regulatory characteristics identified in this study differ from those reported in previous studies on the molecular features of Holstein cattle testis. Developmental transcriptome analysis by Gao et al. [[138]53] revealed spermatogenesis-associated upregulation of ROPN1, FSCN3, TNP2, and SPATA16 in mature Holstein testes. ROPN1 (ropporin 1) is located in the sperm flagella and binds to cAMP dependent protein kinase (PKA)/A-kinase anchoring protein (AKAP), affecting sperm motility, phosphorylation and sheath integrity [[139]53, [140]54]. Down-regulated genes include MORC1 and TMEM119 et al., MORC1 has been demonstrated to be a nuclear protein essential for mouse spermatogenesis, specifically expressed in germ cells [[141]55], and may regulate testicular spermatogenesis by influencing meiotic DNA replication. In contrast, this study provides novel insights into the unique molecular mechanisms of spermatogenesis in Shandong Black cattle testes, employing integrated metabolomic analysis to elucidate a more comprehensive regulatory network. Furthermore, it establishes a theoretical foundation for investigating genetic variations in reproductive performance among different bull breeds. In conclusion, the upregulation of DEPs promotes the cell cycle and DNA replication processes, facilitating spermatogenesis in the testis. Conversely, the downregulation of DEPs via metabolic pathways disrupts germ cell metabolism in the testis, inhibiting spermatogenesis. Integrating and analysing the KEGG and GO enrichment results can help identify candidate markers for spermatogenesis. WGCNA revealed that 1059 DEPs within the representative module"MEturquoise"were significantly and positively correlated with testis weight, including AKAP4 (A-Kinase Anchoring Protein 4). Studies have shown that in AKAP4 knockdown mice, sperm count and motility are reduced, and abnormal flagellar fibre sheaths are observed [[142]56, [143]57]. Sperm motility is positively correlated with testis weight [[144]35–[145]37], indicating that AKAP4 is positively associated with testis weight. After performing GO and KEGG enrichment analyses of the DEPs, we conducted PPI network analysis and identified 14 key regulatory proteins, including MCM3, MCM4, MCM5, MCM6, CDK1, CDK2, CCNB1, CCNB2, CDK7, and CCNH. All these proteins are present in the"MEturquoise"module and are positively correlated with testis weight. CDK1 binds to CCNB1 and CCNB2 to drive spermatocytes into mitosis [[146]23], facilitating spermatogenesis in the testis. CDK7 forms an active complex with CCNH to activate CDK1 and CDK2, thereby participating in the meiotic cell cycle process [[147]28] and influencing spermatogenesis. MCM3 interacts with CDK1 to facilitate the loading of the MCM complex onto chromatin, promoting cell cycle progression and subsequent proliferation of Sertoli cells and Leydig cells [[148]58]. In conclusion, by integrating WGCNA, PPI, and pathway analysis results, MCM3, MCM4, and CDK1 can be considered candidate marker proteins for further investigation in combination with metabolomics data to explore their impact on spermatogenesis in the testis. Differential metabolite analysis showed that 73 differential metabolites between the large and small testis tissues of Shandong black cattle, with 14 upregulated and 59 downregulated. Upregulated metabolites include the sex hormone testosterone. In this study, bulls were in a critical period of sexual maturity, and the increase of testosterone level promoted the rapid proliferation and development of testicular Sertoli cells and Leydig cells [[149]59], which was also consistent with the phenotypic grouping results of this test. The two differential metabolites are mainly related to energy metabolism, including Creatine's participation in the ATP buffer system, which provides energy for sperm movement [[150]60]. Three of differential metabolites are related to oxidative stress, for example, L-Glutathione (reduced) maintains REDOX balance and protects sperm from ROS damage [[151]61]. The other 8 were related to amino acid metabolism and lipid metabolism. Among the metabolites down-regulated: 12 of differential metabolites are related to fatty acid metabolism, such as Elaidic acid, which can impair steroidogenesis and spermatogenesis, inducing sperm head/tail/neck abnormalities, and have toxic effects on testicular tissue by increasing oxidative stress, inflammation and apoptosis [[152]62]. 7 metabolites are related to amino acid metabolism, such as DL-Norvaline, which can interfere with Arginine metabolism [[153]63], thus affecting sperm membrane integrity and mitochondrial potential, as well as the percentage and quality of Arginine capacitated sperm [[154]64]. 12 of differential metabolites are related to nucleotide metabolism, such as hypoxanthine indicated purine metabolism disorders potentially compromising sperm genetic integrity through DNA damage [[155]65]. The remaining 33 differential metabolites were mainly related to carbohydrate and oxidative stress. In conclusion, up-regulated metabolites may promote testicular spermatogenesis by promoting energy metabolism and maintaining REDOX balance. Down-regulated metabolites may affect testicular spermatogenesis through amino acid and nucleotide metabolism disorders. The regulatory mechanism of spermatogenesis in Shandong black cattle could be further clarified by combined analysis with proteomics. Metabolomic KEGG analysis revealed that the significantly upregulated metabolites were predominantly associated with androgen and estrogen receptor agonists/antagonists (e.g., testosterone), Glycerolipid metabolism (uridine diphosphate glucose). In contrast, the significantly downregulated metabolites were enriched mainly in metabolic pathways involving hypoxanthine. Combined metabolomic and proteomic KEGG pathway analysis revealed that uridine diphosphate glucose (UDP-glucose) is a downstream product of glycolysis. Liu et al. [[156]52] demonstrated that proliferating mammalian cells tend to utilize glycolysis to accumulate intermediate metabolites during the S phase of the cell cycle for subsequent DNA replication and cytoplasmic division. The upregulation of UDP-glucose suggests an increased glycolytic energy supply, accelerating the progression of the cell cycle through glycolysis. This upregulation enhances MCM3/MCM4 activity during S phase, promoting the growth and proliferation of Leydig cells and Sertoli cells in the testis and thereby enhancing spermatogenesis (Fig. [157]5). Previous studies have shown that hypoxanthine maintains meiotic arrest by inhibiting phosphodiesterase and thereby blocking the degradation of intracellular cAMP [[158]66, [159]67]. An increase in intracellular cAMP levels triggers the activation of protein kinase A (PKA), which in turn inactivates CDC25B (CDK1-activating factor) and activates Wee1/Myt1 kinases, leading to CDK1 phosphorylation and inactivation of the maturation-promoting factor (MPF, a complex of CDK1 and cyclin B), thereby hindering the G2/M transition of the cell cycle [[160]66–[161]68]. Elevated hypoxanthine levels inhibit CDK1 activity, resulting in a blockade in the G2/M phase of the cell cycle in testicular germ cells, affecting the proliferation and division of Leydig cells and Sertoli cells in the testis, and suppressing spermatogenesis (Fig. [162]5). Immunohistochemical analysis revealed significantly higher expression levels of CDK1, MCM3, and MCM4 in the G1 group testes compared to the G2 group. In G1 testes, CDK1 exhibited strong immunoreactivity predominantly localized in both the cytoplasm and nuclei of Sertoli cells, spermatogonia, primary spermatocytes, and secondary spermatocytes. In contrast, G2 testes showed only weak cytoplasmic staining in Leydig cells, spermatogenic cells at various stages, and Sertoli cells. This distribution aligns with CDK1’s role in cell cycle regulation, and its elevated expression in the G1 group suggests enhanced spermatogenic activity. MCM3/MCM4 were detected in the cytoplasm and nucleus of Leydig cells, spermatogenic cells, and Sertoli cells, with particularly strong signals in Leydig cells, Sertoli cells, and spermatogonia. This pattern implies heightened DNA replication and proliferative activity in these cell types. The G1 group exhibited markedly stronger MCM3/MCM4 expression than the G2 group, potentially indicating more robust spermatogenesis, while the low expression in G2 may reflect cell cycle restriction or arrest. The upregulated expression and distinct localization of CDK1, MCM3, and MCM4 in the G1 group testes support their involvement in cell proliferation and DNA replication, consistent with their regulatory functions in the cell cycle. qPCR analysis revealed that MCM3, MCM4, and CDK1 were expressed in heart, liver, spleen, lung, kidney, muscle, and testis. The expression levels of MCM4 and CDK1 in testicular tissue were significantly higher than in other tissues, with the lowest expression observed in muscle. MCM3 exhibited the highest expression in lung, followed by testis. Western Blot verification further confirmed the expression of MCM3, MCM4, and CDK1 proteins across different tissues. In testicular tissue, both MCM3 and MCM4 showed higher protein expression levels compared to other tissues, and their transcriptional levels were consistent with the protein expression trends. Notably, the transcriptional level of these genes in testicular tissue was higher than their corresponding protein levels. CDK1 expression was highest in lung and kidney but lower in heart, liver, spleen, muscle, and testis, which was generally consistent with the transcriptional trend, except for a decrease in testis expression at the protein level. These results indicate that MCM3, MCM4, and CDK1 are stably expressed at both transcriptional and protein levels, with post-transcriptional modifications influencing protein synthesis. Additionally, histone expression and transcription levels in the G1 group were higher in testis compared to the G2 group, suggesting that the expression of MCM3, MCM4, and CDK1 is positively correlated with testicular weight. This finding aligns with the screening results of the candidate marker proteins. Therefore, MCM3, MCM4, and CDK1 can be considered candidate marker proteins for regulating testicular spermatogenesis. This study integrated proteomic and metabolomic data to preliminarily identify candidate proteins that influence spermatogenesis. It clearly highlighted the benefits of multi-omics integration in overcoming the limitations of single-omics approaches and enhancing research depth. These molecular markers have demonstrated substantial application value in breeding practices, such as utilizing MCM3 protein levels for the early prediction of fertility in breeding bulls. However, the current research still has some limitations. This study used four bulls per group, and although PCA and heat map clustering showed good agreement within the groups, future studies should increase the sample size to improve reproducibility. There is also a lack of in vivo functional experiments to verify the localization and causal effects of these targets.In the future, conditional knockout animal models could be constructed using technologies like CRISPR-Cas9, and single-cell sequencing techniques could be integrated to further elucidate the regulatory mechanisms and application potential of these molecular markers, thereby providing a more robust theoretical foundation and technical support for the genetic improvement of breeding bulls. Conclusions MCM3, MCM4 and CDK1 are involved in regulating spermatogenesis in the testis. Supplementary Information [163]Supplementary Material 1.^ (1.8MB, pdf) Acknowledgements