The epididymis, primarily composed of an epithelial membrane and a thin annular muscle, exhibits secretory functions that contribute to sperm nutrition and maturation. Species variations are observed in the length and morphology of the epididymis. Notably, mice possess an epididymal tube exceeding 1 m in length (approximately 100 times longer than the epididymis itself), while rats have 3 m and humans have 6 m (Von and Neuhaeuser, 1964; Turner et al., 1990; Jiang et al., 1994; Stoltenberg et al., 1998). As a component of the male reproductive tract, the development of the epididymis is dependent on androgens and originates from the Wolffian duct (Shaw and Renfree, 2014). Studies indicated that Hoxa10 and Hoxa11 genes, belonging to the homeobox (Hox) gene family, played crucial roles in epididymal development (Branford et al., 2000; Snyder et al., 2010).

The function of the epididymis is closely related to the sperm maturation. It has been established that mammalian spermatozoa undergo a process of “ripening” as they traverse the epididymis (Westfalewicz et al., 2017) While human sperm transport typically takes an average of 12 days (Rowley et al., 1970). In addition to these maturation steps, the epididymis provides a sophisticated microenvironment that allows for stationary storage of sperm during epididymal transit, avoiding premature sperm activation (Acott and Carr, 1984). Fundamentally, the four primary functions of the epididymis encompass storing, transporting, protecting, and eliminating sperm.

The earliest report on the epididymis was published in the 18th century (National Library of Medicine, 1859): discutient application to the indurated epididymis, focusing on discutient application to treat indurated epididymis. Early understanding of the epididymis stemmed from various pathological studies (Rockwell, 1888; Bryant, 1892; Some Diseases of the Male Genital System, 1908) and investigations into its anatomical structures and functions (Griffiths, 1893; Watson, 1902). Since the 1950s, a significant number of research papers had emerged in this field. Over the past 70 years, extensive exploration had been conducted on different aspects of the epididymis. Initially, pioneers elucidated its anatomy in horses (Goglia, 1954), guinea pigs (Goglia and Magli, 1957) and humans (Montagna, 1952). Subsequently, cellular and chemical analyses gained prominence with cytochemical research and histochemical study of mouse epididymis (Allen, 1961; Allen and Slater, 1961; Birnbaum et al., 1961). Since the 1960s, continuous research efforts were dedicated to study the epididymis as evidenced by an increasing number of annual publications depicted in Figure 1A. With advancements in sequencing technology came a greater emphasis on understanding detailed functions of the epididymis. Consequently, its crucial role in sperm maturation was discovered (Salisbury et al., 1963; Salisbury and Graves, 1963). This led to further investigations into the proteome, transcriptome, and genetics of this organ resulting in numerous reported findings (Dacheux et al., 2009; Wong et al., 2020; Zheng et al., 2021). Transcriptomic and proteomic techniques facilitated progress in studying gene expression specific to the epididymis along with their associated functions (Lye and Hinton, 2004; Légaré and Sullivan, 2020). It is now well-established that each segment within this highly segmented structure expresses distinct genes as well as the related proteins (Domeniconi et al., 2016). Figure 1B illustrated that research pertaining to region-specific gene expression patterns began appearing since 1983 with an upward trend observed annually.

It had been reported that the environment of the epididymal duct might influence on the steps of sperm maturation (Shimazaki et al., 1976). During the process of mammalian sperm transport through the epididymis, the sperm encountered intraluminal fluid with disparate protein compositions, which interacted with the sperm surface and confers fertilization capabilities to the male gamete (Dacheux et al., 2005). Numerous studies had shown that a majority of sperm in the cauda region of epididymis were matured. As sperm passed through the epididymis, they undergo capacitation, acquire fertility, and could be used for in vitro fertilization (IVF) or artificial insemination (AI) (Axnér et al., 1999; Tsutsui et al., 2003; Toyonaga et al., 2011).

Rats and mice had been the most commonly used research models for studying the epididymis. In recent decades, advancements in technology, such as gene-knockout approaches (Eddy et al., 1996), transcriptome sequencing (Wu et al., 2021a), microarray analysis (Jervis and Robaire, 2003), proteomics (Fouchécourt et al., 2000), cell lines (Masuda et al., 2022), and single cell RNA-seq methods for transcriptome analysis (Rinaldi et al., 2020; Lang et al., 2022), had significantly enhanced our understanding of epididymal composition. Numerous knockout mouse models have exhibited phenotypes that impact male reproductive function. For instance, sperm from Clgn, Adam1a, Adam2, and Adam3 knockout mice were unable to migrate to the fallopian tubes (Cho et al., 1998; Ikawa et al., 2001; Nishimura et al., 2004). However, the dynamic functioning of these genes remained largely unknown. Both in vivo and in vitro models had been employed to investigate epididymal function. Currently, culturing techniques for epididymal cells had become well-established (Klinefelter et al., 1982; Chen et al., 1998; Lee et al., 2007). A rat caput epididymal cell line had demonstrated characteristics similar to epithelial principal cells found in vivo (Araki et al., 2002; Dufresne et al., 2005). The advent of RNA sequencing technologies had enabled researchers to analyze the transcriptomes of specific groups or even individual cells (Vazquez et al., 2019). Correlative studies had revealed novel distribution patterns of mitochondria and key genes that might be as-sociated with initial and subsequent sperm waves (Shi et al., 2021).

At present, there are many achievements in the application of single cell technology in the field of epididymis research. Human nasal, bronchial and epididymal epithelial cells were compared by single cell analysis. It has been found that when secretory cells, ciliated cells and basal cells are located in different anatomical locations, their transcriptome features are different (Paranjapye et al., 2022). Eight clusters of cell types have been identified in the proximal human epididymis, including secretory and basal epithelial cells, as well as rare clear cells, all of which have overlapping functions with bronchial and nasal epithelial cells (Paranjapye et al., 2022). Aged principal cells showed a variety of functional gene expression changes related to acrosome response and sperm maturation, suggesting that sperm activation and maturation is an asynchronous process during epididymal transport (Zhuang et al., 2023). At the same time, pathway alterations associated with aging were found in immune cells, particularly “cell chemotaxis” in Cx3cr1Hi epididymal dendritic cells (Zhuang et al., 2023). The monocyte specific expression of chemokine Ccl8 increased with age (Zhuang et al., 2023). Single-cell analysis of zebrafish testis revealed thousands of new marker genes specific to cell types, and through ligand receptor (LR) analysis, it was found that Zebrafish stromal cells had a stronger paracrine effect on germ cells than sertolith cells (Qian et al., 2022). The regulatory network, upstream transcription factors and downstream pathways of human epididymal fragment specific miRNA-mRNA were revealed, which provided a basis for further study of epididymal fragment specific function (Chen et al., 2023). Human epididymis was analyzed by single-cell RNA sequencing to further characterize efferent tubes and model systems (Leir et al., 2023).

The RNA-Seq dataset revealed a substantial number of genes expressed in different regions of the epididymis and the gene expression patterns exhibited significant differences among these regions. Notably, principal cells displayed diverse subsets that were distributed throughout the caput, corpus, and cauda of the epididymis (Rinaldi et al., 2020). However, numbers of genes could be used to distinguish the principal cells from different regions of the epididymal tube. RNase10, Cst11, Lcn2 and Mfge8 genes exhibited high expression levels in caput principal cells. Lcn5, Rnase9 and Plac8 genes were expressed in corpus principal cells (Rinaldi et al., 2020). Additionally, Gpx3, Hint1, Spink10 and Crisp1 showed significant expression in cauda principal cells (Rinaldi et al., 2020). Clear cells were identified by expression of the V-ATPase encoding marker genes such as Atp6v0c, Atpv1e1, and Atp6v1a (Rinaldi et al., 2020). Three clusters of basal cells were identified based on the shared expression of marker genes including Itga6 and Krt14 (Rinaldi et al., 2020). Another study employing single-cell analysis discovered eight subsets within the population of principal cells (Shi et al., 2021). According to GO analysis, two of these subsets were found to be enriched with genes associated with cilia organization and assembly, microtubule-based movement, and cilia-dependent cell movement (Shi et al., 2021). Other major subsets of epididymal epithelial cells were also examined. In detail, four cell subtypes were identified in cluster of basal cells, clear cells, and halo cells (Shi et al., 2021). In this study, the principal cells were characterized by the expression of Aqp9 and Cftr as marker genes, while Krt5 and Cldn1 served as marker genes for basal cells. Clear cells were identified by the presence of Atp6v1b1 and Foxil as marker genes (Shi et al., 2021). Additionally, novel region-specific genes were discovered in principal cells: Lcp1, Pemt, and Ucp2 exhibited high expression levels in the caput; Ccdc198, Ramp3, and Srgn showed high expression levels in the corpus; Atf3 and Zfp36 displayed high expression levels in the cauda of the epididymis (Shi et al., 2021). As new marker genes continue to be discovered, there are slight differences with marker genes in different cell types of different research. It was found that the number of mitochondria in the corpus and cauda of the mice was significantly higher than that in the caput of the epididymis (Shi et al., 2021). Mitochondria are a very important and abundant organelle type in the cytoplasm, involved in the production of adenosine triphosphate (ATP), the establishment of developmental capacity, the maintenance of calcium homeostasis, the regulation of apoptosis and other processes (Shi et al., 2021). The high mitochondrial count and MT% in the epididymis may be required to provide enough energy to synthesize and secrete thousands of molecules that are necessary for various sperm functions (Shi et al., 2021). Over the past 2 years, spatial transcriptomics had provided a wealth of quantitative gene expression data regarding mRNA distribution within tissue slices. These emerging technologies offered new opportunities for bioinformatics analyses in both research and diagnosis (Ståhl et al., 2016). In this paper, we provided an overview of various techniques utilized in epididymis research. Table 1 listed representative research papers showcasing applications of these new technologies within this field. It had been acknowledged that variations in gene and protein expression exist among distinct regions of the epididymis (Johnston et al., 2005), indicating diverse roles or functions in sperm maturation across these regions.

In previous studies, the epididymis had been traditionally classified into three regions–caput, corpus, and cauda. However, in rodents, the epididymis was further divided into four anatomical regions-initial segment, caput, corpus, and cauda (Gervasi and Visconti, 2017). While these divisions were well-established in rodents, they had not been clearly defined in the human epididymis. Previous reports had primarily used the caput, corpus, and cauda as boundaries for analyzing gene and protein expression patterns within the epididymis (Jervis and Robaire, 2001; Chauvin and Griswold, 2004). The caput, corpus, and cauda of the epididymis were proximal to distal, relative to the testis (Figure 2A).

Anatomical analysis of the human epididymis based on histology, proteomics data and 3D reconstruction had led to a subdivision of the caput, corpus and cauda into four six and ten regions respectively (Zhao et al., 2020). This research revealed significant variations in protein composition within different luminal regions of the epididymis (Dacheux et al., 2006). It should be noted that regional divisions of the epididymis also differ among other species. For instance, the mouse epididymis consisted of ten distinct segments while rats comprised fourteen segments (Adamali et al., 1999; Johnston et al., 2005). Furthermore, variations could be observed in size and shape of specific segments acrossing different species (Jelinsky et al., 2007).

The function of the epididymal corpus and cauda were found to be similar. Specifically, immune-related biological processes were enriched in both regions. During transport between the corpus and cauda of the epididymis, sperm acquired fertilization capacity. Currently, it had been demonstrated that sperm in the epididymal corpus area possess a similar ability to cauda sperm, including capacitation, oocyte fertilization, and survival during cryopreservation (Kunkitti et al., 2015; Kunkitti et al., 2016a; Kunkitti et al., 2016b). The cauda of the epididymis served as the final storage site for matured sperm prior to ejaculation (Souza et al., 2020). In many animal species, sperm could maintain fertility for several weeks within this environment (Esponda and Bedford, 1986). These findings highlighted the crucial role of processing and modification in each region of the epididymis for maintaining sperm function.

The biosynthesis of secreted proteins in the cauda of the epididymis, similar to that in the caput, was also regulated by androgen (Foldesy and Bedford, 1982; Wong et al., 1982; Esponda and Bedford, 1986; Regalado et al., 1993). In comparison to other mammals, human sperm production was relatively lower. Furthermore, the functional significance of temperature variation within the epididymis remained unknown (Bedford, 2015). In summary, sperm maturation was intricately linked to the unique regional functions within the epididymis, enabling successful fertilization.

The epididymis comprises convoluted tubules that connect the testis and vas deferens within the male reproductive system. It plays a pivotal role in regulating sperm maturation and fertilization processes. Sperm maturation necessitated epididymal processing, encompassing regional alterations in spermatozoa structure and molecular composition (Jones et al., 2007). Additionally, it involves immune and metabolic physiological mechanisms. Notably, numerous macrophages were present within the epididymal lumen and were capable of recognizing and eliminating suboptimal spermatozoa. Consequently, the epididymis assumed a critical function in both sperm maturation and the acquisition of fertilization competence (Elbashir et al., 2021).

With the advancement of sequencing technologies (Wong et al., 2020; Wu et al., 2021a; Zhao et al., 2021a; Liu et al., 2022b), a greater understanding of the molecular mechanisms underlying epididymal function has been achieved. The significance of the epididymis in male reproductive biology has garnered increasing attention. Furthermore, distinct regional disparities within the epididymis have been elucidated (Belleannée et al., 2012; Cyr et al., 2018; Wu et al., 2021b; Zhao et al., 2021b). For instance, the caput region exhibited heightened activity and accounted for approximately 70%–80% of total protein secretion (Cornwall, 2009). The corpus of the epididymis plays a pivotal role in immune functions. The comprehension of the epididymis evolved from its initial morphological and structural characterization to encompass diverse functional aspects across different regions (Martin et al., 1902; Sullivan et al., 2019; James et al., 2020; Zhao et al., 2020). Notably, unique gene expression patterns were identified in the caput, corpus, and cauda regions through techniques such as microarray or single-cell RNA sequencing analysis. In summary, while the caput region secretes nutrients and androgens to facilitate sperm maturation, sperm traverse through the corpus region towards the cauda, which acts as a barrier against autoimmune reactions, additionally serving as a conduit for transporting sperm to the vas deferens. Consequently, functional discrepancies exist among various regions within the epididymis due to differential gene expression profiles that drive specific functions associated with each respective region.

The male reproductive system exhibited significant interspecies variability. In the epididymis of rats and mice, distinct regions displayed pronounced differences (Abou-Haïla and Fain-Maurel, 1984). Due to their shorter life cycle than humans, rodents have become the most common animal model in research. Recently, dogs have emerged as viable biological models for studying the molecular functions of the epididymis (Kirchhoff, 2002). However, extrapolating reproductive characteristics of other mammals based solely on a specific species might lead to biased conclusions in terms of reproductive biology.

The cell classifications of the epididymal epithelium in each region of the epididymis varied (Chen et al., 2021). Among them, the principal cells constituted 80% of the epithelial cells in the epididymis tubule. Further analysis revealed distinct gene expression patterns for the same cell type across different regions of the epididymis. In this study, we reanalyzed and visualized gene expression data from the mouse epididymis and vas deferens based on previous findings and publicly available single-cell datasets. Marker genes specific to caput principal cells included RNase10, Cst11, and Lcn2. Similarly, the Lcn5, RNase9, and Plac8 genes distinguished corpus principal cells, while the Gpx3, Klk1b27, Hint1, and Gstm2 genes characterized cauda principal cells. Basal cells were identified by the Itga6 and Krt14 genes. Interestingly, our analysis revealed differential mRNA expression levels between principal and basal cells for several genes, including Lcn5, which showed high expression in caput principal cells but was significantly detected in both caput and corpus basal cells; Defb28 exhibited high expression in cauda principal cells but was significantly detected in epididymis basal cells as well (Rinaldi et al., 2020). The results demonstrated the essential role of Rnase10 in sperm adhesion within the epididymis, a critical characteristic for mouse sperm transport in the female vagina (Krutskikh et al., 2012). Combined with the data analysis on Lcn5 in the principal cells, we indicated that the caput of the mouse epididymis was related to lipid metabolism and adhesion. Similarly, basal cells exhibited significant enrichment of genes related to membrane transport and lipid metabolism (Rinaldi et al., 2020). These findings suggested that different cell types share similarities, contributing to an optimal environment for sperm maturation within the epididymal cavity. Additionally, certain coregulatory factors displayed fragment-specific enrichment patterns during their expression processes, suggesting their potential contribution to specific responses (Browne et al., 2016). Each region of the epididymis possesses a unique transcriptomic signature, which regulates luminal composition and ultimately influences male sperm maturation.

The intricate patterns of gene expression and cellular composition in the caput, corpus, and cauda regions of the epididymis contribute to distinct functions in sperm maturation. However, interspecies variations exist in gene expression within the epididymis. Exploring the functional diversity across different regions of the epididymis presents an exciting avenue for research. It is anticipated that advancements in sequencing technologies, such as spatial transcriptomics, would play a pivotal role in unraveling the complexities of epididymal biology.