Region-specific transcriptomic responses to CD52 deficiency in the male reproductive tract

Abstract

Background

Spermatozoa are produced in the testis and acquire motility and fertilizing capacity during post-testicular maturation in the epididymis, a highly region-specific process. How gene loss reshapes region-specific transcriptional programs along the testis–epididymis axis remains poorly defined, particularly in the absence of overt fertility defects. CD52 is expressed in male reproductive tissues, yet whether CD52 loss elicits region-specific transcriptomic alterations along the male reproductive tract remains unclear. In this study, we aimed to systematically characterize spatially resolved transcriptomic changes associated with CD52 deficiency across the testis and epididymal segments.

Results

CD52-knockout male mice displayed normal fertility, as assessed by successful mating and offspring production, and showed no obvious histological abnormalities in the testis or epididymis. Multi-region bulk RNA sequencing identified differentially expressed genes in the testis (119), caput epididymis (284), corpus epididymis (577), and cauda epididymis (294). Functional enrichment analyses revealed that pathways related to cytoskeletal organization and motor protein function were predominantly affected in the testis, caput, and cauda. In contrast, pathways associated with cellular homeostasis and ion transport were uniquely enriched in the corpus epididymis. Protein–protein interaction network analysis further identified region-specific hub genes, including Ttn in the testis and cauda, Tcap in the caput epididymis, as well as Spp1 in the corpus epididymis. Comparative analyses between adjacent tissues highlighted pronounced transcriptional heterogeneity along the testis–epididymis axis in response to CD52 deficiency.

Conclusion

This study provides a region-resolved transcriptomic characterization of CD52 deficiency across the testis and epididymal segments. Our findings demonstrate that CD52 loss is associated with distinct, spatially organized transcriptional responses along the male reproductive tract, offering insight into region-dependent molecular regulation in the absence of overt reproductive phenotypes.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12652-8.

Introduction

The production of functional male gametes is a complex process that involves spermatogenesis in the testes, followed by post-testicular maturation in the epididymis [1, 2]. Spermatogenesis is a highly coordinated process regulated by precise stage and cell-type-specific gene expression programs within the testis [3, 4]. Spermatozoa leaving the testis are not fully competent and undergo progressive acquisition of motility and fertilizing capacity during transit through the epididymal duct [5–7]. The epididymis is anatomically and functionally segmented into the caput, corpus, and cauda, each characterized by distinct epithelial environments and region-specific gene expression profiles [8–11]. Functional studies indicate that the caput and corpus are primarily involved in early and late maturation processes, respectively, whereas the cauda serves as a storage site for mature spermatozoa [12]. The testis and epididymis constitute a continuous and highly regulated transcriptional axis, providing a valuable in vivo framework for investigating spatial regulation of gene expression underlying sperm development and maturation.

Gene knockout models are widely used to investigate the biological functions of specific genes in male reproduction by comparing testicular and epididymal development, spermatogenesis, and fertility between wild-type (WT) and mutant animals [13, 14]. While deletion of many genes essential for spermatogenesis leads to embryonic lethality or male infertility [15–18], there are also numerous cases in which genetic ablation of testis- or epididymis-enriched genes does not result in overt reproductive phenotypes [19–24]. Importantly, the absence of obvious phenotypic defects does not preclude the presence of underlying molecular or transcriptional alterations. In this context, transcriptome-based analyses across multiple reproductive tissues provide a complementary approach to uncover region-specific molecular responses to gene deletion that may not be evident at the phenotypic level.

CD52 is a glycosylphosphatidylinositol-anchored protein that is widely expressed in immune cells and has also been detected in the male reproductive tract, predominantly in the epididymis [25, 26]. In humans, CD52 expression has been positively correlated with sperm motility, whereas reduced expression has been observed in individuals with oligozoospermia and asthenozoospermia [27]. CD52 has also been suggested to be associated with sperm maturation and immunological modulation within the epididymal environment [25], and has been proposed as a sorting marker for human epididymosomes [28]. Notably, previous studies have reported that CD52-deficient mice generated by homologous recombination do not exhibit overt defects in tissue morphology and male fertility under physiological conditions. However, the molecular consequences of CD52 deficiency in reproductive tissues, particularly in a region-resolved manner along the male reproductive tract, remain poorly characterized.

In this study, we generated a CD52-knockout (CD52-KO) mouse model using CRISPR/Cas9 and performed a multi-region transcriptomic analysis of the male reproductive tract. RNA sequencing was conducted on the testis and three epididymal segments (caput, corpus, and cauda) from WT and CD52-KO male mice. By integrating comparative transcriptomic analyses with functional enrichment and protein–protein interaction (PPI) network analyses, we systematically characterized region-specific transcriptional responses associated with CD52 deficiency. This study provides a comprehensive transcriptomic resource that highlights spatial heterogeneity along the testis–epididymis axis, thereby offering a framework for understanding region-specific regulatory alterations in the male reproductive tract in the absence of overt reproductive abnormalities.

Materials and methods

Ethics statement

The use and care of animals complied with the guidelines of the Biomedical Research Ethics Committee of the Agricultural Genomics Institute in Shenzhen, Chinese Academy of Agricultural Sciences. Mice were maintained under a 12-hour light/dark cycle at a constant room temperature of 21–23 °C in ventilated cages with ad libitum access to food and water.

Generation of the CD52-KO mice

CD52-KO mice were generated using a CRISPR/Cas9-mediated genome editing approach, as previously described [29]. Briefly, superovulation was induced in 12–15 female C57BL/6J mice (3–4 weeks old) by intraperitoneal injection of pregnant mare serum gonadotropin (PMSG, 5 IU), followed 48 h later by human chorionic gonadotropin (hCG, 5 IU). After hCG administration, females were housed overnight with C57BL/6J males. On the following morning, females with vaginal plugs were identified and used for zygote collection. Zygote–cumulus complexes were treated with hyaluronidase to remove surrounding cumulus cells, washed several times in M2 medium, and cultured in KSOM medium at 37 °C in a humidified incubator with 5% CO₂.

Two single-guide RNAs (sgRNAs) targeting exon 2 of the CD52 gene were designed using the CHOPCHOP web tool (https://chopchop.cbu.uib.no/) based on predicted on-target efficiency and minimal off-target potential (Supplementary Table S1). A mixture containing Cas9 mRNA (100 ng/µL) and synthesized sgRNAs (50 ng/µL for each sgRNA) was microinjected into approximately 80 fertilized zygotes. Injected embryos were cultured in KSOM medium for approximately 2.5 days until the blastocyst stage. The resulting blastocysts were subsequently transferred into the oviducts of pseudopregnant ICR recipient females, with 15–20 embryos transferred per recipient. A total of 12 founder mice carrying the intended deletion were obtained.

Genomic DNA was extracted from the tail or foot of the resulting pups. Genotyping was performed using polymerase chain reaction (PCR) and Sanger sequencing to confirm the edit in CD52. A 190-bp deletion was introduced in exon 2 of the CD52 gene, which was validated by PCR and Sanger sequencing and is predicted to cause a frameshift mutation that disrupts the CD52 coding sequence. The overall workflow of mouse generation and subsequent breeding is illustrated in Supplementary Figure S1.

Breeding strategy

Male CD52-KO founder mice were initially crossed with WT females to generate heterozygous offspring. These heterozygous mice were subsequently backcrossed with WT animals for at least three generations. Heterozygous males and females were then intercrossed to obtain homozygous CD52-KO mice, which were maintained through littermate intercrosses for an additional three generations before experimental use. Genotype distributions among offspring followed expected Mendelian ratios. When multiple eligible male littermates of the same genotype were available, animals were randomly selected for downstream analyses.

Tissue collection

Testis and epididymal segments (caput, corpus, and cauda) were collected simultaneously from age-matched (10-week-old) C57BL/6J WT and CD52-KO male mice (n = 4 per group). For downstream analyses, tissues were processed in parallel for either histology or transcriptomic profiling, as described below.

Histology

For histological analysis, freshly dissected tissues were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin (H&E) following standard protocols. Specimen sectioning and staining were done by Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). Images were captured using a Leica bright-field microscope system and viewed with NDP.view 2 software.

RNA extraction and transcriptomic analysis

For transcriptomic analysis, tissue samples were snap-frozen in liquid nitrogen and stored at − 80 °C until RNA extraction. All transcriptomic library preparation and sequencing were performed by GENEWIZ, Inc. (Suzhou, China). mRNA libraries were generated through poly(A) mRNA enrichment, RNA fragmentation, reverse transcription using random hexamers or oligo (dT) primers, adaptor ligation, and PCR amplification. High-throughput sequencing was carried out on the DNBSEQ-T7 platform; one corpus epididymis sample failed and was excluded. RNA-seq data generated in this study are available in the NCBI BioProject database under accession number PRJNA1257005 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1257005). Sample IDs (e.g., mus-WT-T1) correspond to the names of the raw data files. Abbreviations: T = testis, H = caput epididymis, EP = corpus epididymis, TA = cauda epididymis.

Raw sequencing reads were assessed for quality using FastQC (version 0.11.2) and then processed with fastp (version 0.22.0) to remove adaptor sequences and low-quality reads [30]. Clean reads were aligned to the mouse reference genome (GRCm39) using STAR (v2.7.10b) [31]. Gene-level read counts were obtained using RSEM (v1.3.1) [32].

Differential gene expression analysis was performed using the DESeq2 package (v1.45.0) [33]. DESeq2 normalization and dispersion estimation were performed using the default DESeq2 pipeline. Differential expression testing was conducted using the Wald test, and p-values were adjusted for multiple testing using the Benjamini–Hochberg false discovery rate (FDR) method. Genes with an adjusted p-value < 0.05 and |log2(fold change)| > 1 were considered differentially expressed. Lowly expressed genes were subject to independent filtering within the DESeq2 framework during multiple testing correction. All samples were processed under uniform experimental conditions. Differential expression analysis was performed using a design formula including genotype as the primary factor (design = ~ genotype, with WT and CD52-KO defined as the two genotype groups).

For Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, all expressed genes detected in the corresponding tissue were used as the background gene set. Enrichment analyses were performed using the clusterProfiler package (v4.8.3) [34]. All statistical analyses and data visualization were conducted using R (v4.3.1).

Protein–Protein Interaction (PPI) analysis

PPI networks for DEGs were constructed using the STRING database (v12.0) (https://string-db.org/) [35] with a confidence score threshold > 0.4. Network visualization was performed using Cytoscape (v3.10.2), and hub genes were identified using the cytoHubba plugin [36].

Results

Generation of CD52 knockout mice models and reproductive tissue morphology

We generated CD52 knockout mice by introducing a 190-bp deletion in exon 2 using CRISPR/Cas9, and homozygous KO mice were selected for subsequent experiments (Fig. 1A, Supplementary Fig. S2A–C).

Fig. 1.

Generation and tissue morphology evaluation of reproductive tissues in WT and CD52-KO male mice. A Schematic diagram of the CRISPR/Cas9-mediated strategy for CD52 gene knockout. Two sgRNAs, sgRNA1 and sgRNA2, were designed to target exon 2, resulting in a 190 bp deletion (red indicates the sgRNA sequences, green indicates the PAM sequences). Arrows indicate primer positions (F, forward; R, reverse) used for genotyping. B Representative H&E-stained tissue sections showing testis and epididymis morphology in WT and CD52-KO mice. Bars, 50 µm. C Bar plots showing CD52 expression levels in the testis and three regions of the epididymis (caput, corpus, and cauda) from WT (red) and CD52-KO (blue) mice based on RNA-seq data. Expression values represent DESeq2-normalized counts

Histological examination of H&E-stained sections revealed no major morphological differences between WT and CD52-KO mice across the testis, caput, corpus, and cauda epididymis (Fig. 1B). The overall epithelial structure and luminal organization were well preserved in both groups.

Sequencing depth and genome mapping rates were examined for all four reproductive tissues (Supplementary Figure S3A–D). Each sample generated approximately 38–55 million reads, with consistently high mapping rates above 85% across both WT and CD52-KO groups, confirming that the RNA-seq libraries were of high quality and suitable for downstream analyses.

In WT mice, CD52 expression was markedly higher in all epididymal regions compared with the testis, with the strongest expression observed in the cauda region. As expected, CD52 expression was nearly undetectable across all four tissues in CD52-KO mice (Fig. 1C), confirming the successful knockout of the gene in these tissues.

Transcriptomic alterations in the testis

Transcriptomic profiling of four tissues, including the testis and three epididymis regions (caput, corpus, and cauda), was performed in WT and CD52-KO mice. For the testis analysis, principal component analysis (PCA) revealed a clear separation between the two groups, indicating distinct global transcriptomic profiles (Fig. 2A). Volcano plots visualized the scale and direction of expression changes between groups, including 72 upregulated and 47 downregulated genes in the CD52-KO group compared to WT controls (Fig. 2B). Hierarchical clustering based on the expression of DEGs also demonstrated a clear distinction between WT and CD52-KO samples, as shown in the heatmap (Fig. 2C).

Fig. 2.

Transcriptomic analysis of the testis in WT and CD52-KO mice. A PCA of gene expression profiles from WT (red) and CD52-KO (blue) testis samples. B Volcano plot illustrating the distribution of DEGs between groups (threshold: |log₂FC| > 1 and adjusted p-value < 0.05). Red and blue dots represent significantly upregulated and downregulated genes in CD52-KO mice, respectively. C Heatmap showing hierarchical clustering of DEGs in WT and CD52-KO testes. D GO enrichment analysis of DEGs, categorized by biological process (BP), cellular component (CC), and molecular function (MF). E KEGG pathway enrichment analysis showing pathways associated with the identified DEGs. Dot size indicates the number of genes; color reflects −log10(q-value)

GO enrichment analysis revealed significant enrichment in terms related to cytoskeletal structure and function, including muscle system process, supramolecular fiber, myofibril, actin binding, and structural constituent of cytoskeleton (Fig. 2D). Additionally, KEGG pathway analysis further highlighted enrichment in cytoskeleton in muscle cells and motor proteins pathway (Fig. 2E). These findings showed that CD52 deficiency alters gene expression programs related to cytoskeletal organization and contractile function in the testis.

Transcriptomic alterations across epididymal segments

To examine the impact of CD52 deficiency on the epididymis, we performed transcriptomic profiling of three major regions: the caput, corpus, and cauda epididymis. PCA showed partial separation and genotype-associated clustering trends between WT and CD52-KO samples across all three epididymal segments (Supplementary Figure S4A, D, G), suggesting genotype-associated transcriptomic differences. Volcano plots visualized the scale and direction of expression changes between groups (Supplementary Figure S4B, E, H). Differential expression analyses identified 284 DEGs in the caput (137 upregulated and 147 downregulated), 577 DEGs in the corpus (301 upregulated and 276 downregulated), and 294 DEGs in the cauda (85 upregulated and 209 downregulated), indicating region-specific gene expression changes across all epididymal segments upon CD52 deletion. Hierarchical clustering based on the expression of DEGs also demonstrated a clear distinction between WT and CD52-KO samples, as shown in the heatmaps (Supplementary Figure S4C, F, I).

In the caput, GO enrichment analysis revealed significant associations with actin binding, supramolecular fiber, and actin cytoskeleton organization (Supplementary Figure S5A). KEGG analysis further showed enrichment in pathways such as cytoskeleton in muscle cells and motor proteins (Supplementary Figure S5B). In the corpus, GO terms were enriched for cellular homeostasis, supramolecular polymer, and DNA-binding transcription repressor activity (Supplementary Figure S5C). While pathways included cellular senescence, FoxO signaling, and Apelin signaling pathway were enriched in the Corpus epididymis (Supplementary Figure S5D). The cauda showed a functional profile partially overlapping with the caput, with enrichment of GO terms such as actin filament-based process, supramolecular fiber, calcium ion binding, and organelle assembly (Supplementary Figure S5E). Corresponding KEGG analysis included the cytoskeleton in muscle cells and motor proteins (Supplementary Figure S5F).

Taken together, these results demonstrate that CD52 deficiency alters the transcriptomic landscape across the epididymis in a region-specific manner. The caput and cauda epididymis exhibited similar enrichment patterns related to cytoskeletal structure and dynamics, whereas the corpus epididymis exhibited a distinct transcriptional profile dominated by cellular homeostasis–associated processes.

PPI network analysis

PPI networks were constructed for each tissue, and hub genes were identified based on degree centrality (Fig. 3A–D). In both the testis and cauda epididymis, Ttn (Titin) was identified as the hub gene (Fig. 3A, D). The caput epididymis network highlighted Tcap (Telethonin, also known as Titin-cap) as the hub gene (Fig. 3B). The corpus epididymis displayed a distinct PPI pattern centered on Spp1 (Secreted phosphoprotein 1) (Fig. 3C). Consistent with the enrichment results, Ttn and Tcap were central nodes in networks enriched for cytoskeleton- and motor protein-related pathways, while Spp1 occupied a central position in networks linked to cellular homeostasis and cell senescence pathways.

Fig. 3.

PPI network and hub gene expression in the testis and epididymis segments. PPI networks constructed from DEGs in the testis (A), caput epididymis (B), corpus epididymis (C), and cauda epididymis (D). Node size reflects interaction centrality; hub genes are centrally located. Expression of representative hub genes in WT (red) and CD52-KO (blue) mice: (E) Ttn in the testis, (F) Tcap in the caput, (G) Spp1 in the corpus, and (H) Tcap in the cauda. Error bars represent the standard error of the mean (SEM)

Expression analysis further confirmed these patterns (Fig. 3E-H). Ttn and Tcap were downregulated in the CD52-KO group, while Spp1 was specifically upregulated in the corpus epididymis. These results highlight the region-specific regulatory programs in response to CD52 loss, with the testis, caput, and cauda showing convergent enrichment of cytoskeleton-related gene expression patterns, whereas the corpus exhibited a distinct transcriptional profile associated with cellular homeostasis-related processes.

Region-specific transcriptomic divergence along the testis–epididymis axis

Building upon the DEG and enrichment analyses performed for each tissue individually, we next compared DEGs between adjacent regions along the testis–epididymis axis to evaluate spatial transcriptional coordination under CD52 deficiency. Venn diagram analysis showed that some DEGs were shared between neighboring segments, most were unique to a given region, indicating both common and region-specific transcriptomic responses to CD52 loss (Fig. 4A–C). Specifically, the testis and caput epididymis shared 56 DEGs (16.2%), the caput and corpus shared 67 genes (8.4%), and the corpus and cauda epididymis shared 58 genes (6.8%). These findings support the view that each segment of the testis–epididymis axis exhibits distinct transcriptional alterations in response to CD52 deficiency, consistent with the segment-specific functional enrichment and hub gene profiles identified in earlier analyses.

Fig. 4.

DEGs between adjacent segments of the testis–epididymis axis. Venn diagrams showing the DEGs between adjacent reproductive regions: A testis and caput epididymis, B caput and corpus epididymis, C corpus and cauda epididymis. Each number represents the count of unique or shared DEGs in the corresponding tissue pair

Discussion

The testis and epididymis play essential and regionally specialized roles in male fertility by coordinating spermatogenesis, sperm maturation, and sperm storage through tissue-specific gene regulation programs. CD52 has been reported to be expressed in reproductive tissues, however, the region-specific transcriptomic consequences of CD52 deficiency along the male reproductive tract have not been systematically investigated. In this study, we used CRISPR/Cas9 to generate a CD52 knockout mouse model and performed a multi-region transcriptomic analysis of the testis and three major epididymal segments. Comparative analyses revealed that CD52 deficiency was associated with changes in gene expression across all four tissues. Notably, the testis, caput, and cauda epididymis exhibited similar pathway enrichment patterns, whereas the corpus epididymis displayed a distinct transcriptional profile. Moreover, comparisons of differentially expressed genes between adjacent tissues identified both shared and segment-specific changes, indicating spatially heterogeneous transcriptional responses to CD52 deficiency along the testis–epididymis axis.

CD52 deficiency was associated with transcriptional changes of cytoskeletal, motor protein, and myofibril-related gene sets in the testis, caput, and cauda epididymis. The cytoskeleton, composed of actin filaments, microtubules, and associated proteins, provides the structural framework for intracellular transport and cellular morphogenesis [37]. Motor proteins such as dyneins, kinesins, and myosins generate directional force along these cytoskeletal structures, enabling cargo transport and dynamic cellular remodeling [38–40]. In the testis, actin-based cytoskeletal remodeling is essential for germ cell migration across the seminiferous epithelium, and its disruption can impair spermatogenesis [41–43]. In the epididymis, particularly in the caput and cauda regions, cytoskeletal dynamics support luminal flow, vesicle-mediated secretion, and the establishment of a microenvironment necessary for sperm maturation. Actin-binding proteins, including motor proteins, are key regulators of these processes and have been implicated in both germ cell development and sperm functional maturation [37, 44–46].

PPI network analysis further identified shared hub genes in the testis, caput, and cauda epididymis following CD52 deletion. Specifically, Ttn emerged as a central node in both the testis and cauda, while Tcap was the dominant hub in the caput. Both genes are core components of the sarcomeric cytoskeleton and play roles in stabilizing actin–myosin assemblies and maintaining mechanical tension [47, 48]. Consistent with these structural roles, Ttn has been implicated in spermatogenesis and sperm individualization [49], while Tcap regulates desmin cytoskeleton organization, with its absence leading to cytoskeletal disruption and mitochondrial dysfunction [50]. The identification of these genes as hub nodes aligns with the enrichment observed in this study. These findings suggest that CD52 deficiency is associated with transcriptional remodeling of cytoskeleton-related networks in multiple regions of the male reproductive tract.

Unlike the other three tissues, the corpus epididymis exhibited a distinct transcriptomic profile in response to CD52 deletion, with differentially expressed genes enriched for ion transport, epithelial signaling, and cellular homeostasis-related processes. Consistent with this functional profile, PPI network analysis identified Spp1 as the central hub gene in this segment. SPP1 is broadly expressed across the male reproductive system and has been implicated in the regulation of epididymal epithelial and luminal environments [51]. Notably, previous studies have reported pronounced regional specificity of SPP1 expression within the epididymis across species. In humans, SPP1 is highly expressed in caput epididymal epithelial cells and is androgen-regulated, supporting a role in epithelial-mediated regulation of the epididymal lumen [52]. Similarly, in mouse models with disrupted epididymal signaling, such as HE6/Gpr64 knockout mice, Spp1 expression is downregulated in the caput despite preserved tissue morphology [53]. In contrast, studies in dromedary camels have reported higher Spp1 expression in the corpus epididymis, with corpus-enriched expression positively associated with semen quality in high-fertility individuals. This association has been proposed to involve effects on calcium homeostasis and maintenance of the luminal microenvironment during post-testicular sperm maturation [54]. In addition, Spp1 and CD52 have been reported as co-expressed hub genes in calcific aortic valve disease [55], but their regulatory relationship remains unclear. Accordingly, the present data do not establish a direct mechanistic link between CD52 and Spp1. Further targeted functional studies, including cell-type–specific perturbations or combinatorial genetic approaches, will be required to clarify the biological relevance of their potential interaction in the epididymis.

Region-specific gene expression programs along the testis–epididymis axis reflect the distinct functional roles of each tissue in sperm development and maturation [9]. In this study, we demonstrate that CD52 deficiency is associated with region-specific transcriptional changes across the testis, caput, corpus, and cauda epididymis, highlighting spatial heterogeneity in molecular responses to genetic perturbation. This spatial pattern of transcriptomic divergence is conceptually consistent with recent observations of dynamic small RNA redistribution during post-testicular sperm maturation, in which sperm RNA profiles are transiently remodeled in the caput and partially restored in the cauda [56]. Our study does not directly assess small RNAs or RNA cargo transfer; this parallel highlights shared principles of spatially coordinated and segment-specific regulation along the male reproductive tract. These findings underscore the importance of spatial context in transcriptomic responses to CD52 deficiency along the male reproductive tract. Future validation studies should therefore take into account the region-dependent expression of CD52 when interpreting tissue-specific molecular changes.

CD52 deficiency resulted in transcriptomic alterations across the testis and epididymal segments, while CD52-knockout male mice did not exhibit overt abnormalities in reproductive tract morphology and maintained fertility under physiological conditions. These findings are consistent with previous studies using CD52-deficient mice generated by homologous recombination, which also reported no overt reproductive defects [25]. The maintenance of fertility despite transcriptional changes suggests that compensatory or buffering mechanisms may operate within the male reproductive tract. Similar observations have been reported for other non-essential reproductive genes, where genetic disruption results in molecular or transcriptomic alterations without overt impairment of fertility or reproductive tract morphology [57, 58]. We therefore propose that the preserved fertility observed in CD52-deficient mice may reflect a combination of functional redundancy within regulatory networks and tissue-level robustness of the male reproductive tract. Such mechanisms could buffer molecular perturbations and maintain epithelial organization and luminal homeostasis despite the transcriptomic alterations. Future studies employing targeted functional perturbation, pathway-level analysis, or assessment of luminal composition will be required to test these compensatory mechanisms.

This study has several limitations. First, the transcriptomic alterations identified here were not experimentally validated at the RNA or protein level using approaches such as quantitative PCR, Western blotting, or immunostaining for representative hub genes. Second, bulk RNA sequencing captures average transcriptional changes across heterogeneous cell populations, and it lacks cell-type resolution and temporal information. Given the cellular complexity of the testis and epididymis [59, 60], single-cell or spatial transcriptomic approaches will be required to identify the specific cell populations most responsive to CD52 deficiency. In addition, functional assays, including sperm motility, capacitation, and fertilization efficiency, will be necessary to determine whether the observed transcriptomic changes have physiological consequences under specific conditions. Despite these limitations, this work established a CD52 knockout mouse model using CRISPR/Cas9 and provides a comprehensive, region-resolved transcriptomic analysis of CD52 deficiency across the male reproductive tract.

Conclusion

In summary, using a CRISPR/Cas9-mediated CD52-knockout mouse model, we systematically characterized the transcriptomic responses across the testis and three epididymal segments (caput, corpus, and cauda). CD52 deficiency altered gene expression in all four tissues, with the testis, caput, and cauda epididymis showing coordinated enrichment in cytoskeletal and motor protein pathways, whereas the corpus displayed a distinct transcriptional profile associated with ion transport and tissue homeostasis. These region-specific responses highlight the spatial heterogeneity of gene regulation along the testis–epididymis axis. Collectively, our findings provide a region-resolved transcriptomic framework for understanding molecular responses to CD52 deficiency in the male reproductive tract.

Authors’ contributions

HK: Conceptualization, Methodology, Formal analysis, Software, Resources, Data curation, Writing original draft. HK and YZ: Feed animals, Sample collection. FC and HW: Histology analysis, Review, and editing. MG: Supervision, Review, and editing. GH: Methodology, Software, Review, and editing. LH and RC: Methodology, Review and editing, Supervision, Resources.

Funding statement

This research was funded by Shenzhen Municipal Science and Technology Major Projects (KJZD20230923115003006), the Outstanding Talents Training Fund in Shenzhen, Science and Technology Program of Shenzhen (CJGJZD20210408092402006).

Data availability

RNA-seq data generated in this study are available in the NCBI BioProject number PRJNA1257005 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1257005).

Declarations

Ethics approval and consent to participate

The C57BL/6J mice used in this study were obtained from Cyagen Biosciences Inc. (Guangzhou, China). The use and care of animals complied with the guidelines of the Biomedical Research Ethics Committee of the Agricultural Genomics Institute in Shenzhen, Chinese Academy of Agricultural Sciences.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.