The epididymis-specific proteome
The epididymis functions mainly as a reservoir for sperm and a place where newly synthesized sperm from the testis will acquire their ability to swim and, most importantly, to penetrate the ovum and fertilize the egg. These functions are accomplished by the secretion of several key proteins by the epididymal epithelial cells to create an interactive and dynamic microenvironment in the fluid where the spermatozoa can stay protected and fully mature during their passage through the convoluted tube. Transcriptome analysis shows that 65% (n=13069) of all human proteins (n=20162) are expressed in the epididymis and 315 of these genes show an elevated expression in the epididymis compared to other tissue types.
The epididymis transcriptome
Transcriptome analysis of the epididymis can be visualized with regard to the specificity and distribution of transcribed mRNA molecules (Figure 1). Specificity illustrates the number of genes with elevated or non-elevated expression in the epididymis compared to other tissues. Elevated expression includes three subcategory types of elevated expression:
Distribution, on the other hand, visualizes how many genes have, or do not have, detectable levels (nTPM≥1) of transcribed mRNA molecules in the epididymis compared to other tissues. As evident in Table 1, all genes elevated in epididymis are categorized as:
Figure 1. (A) The distribution of all genes across the five categories based on transcript specificity in epididymis as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (nTPM≥1) in epididymis as well as in all other tissues.
Table 1. The number of genes in the subdivided categories of elevated expression in epididymis.
Protein expression of genes elevated in epididymis
In-depth analysis of the elevated genes in epididymis using antibody-based protein profiling allowed us to visualize the expression patterns of these proteins in different functional compartments including proteins related to innate immune response and host defense, but also sperm maturation and protection.
The first line of defense against invading pathogens is the antimicrobial proteins. A vast majority of these proteins are produced and secreted by immune cells, phagocytic and dendritic cells, but also by all cavity-lining epithelial cells in the body. The beta defensin gene family consists of diverse, antimicrobial and antiviral peptides that have evolved through repeated gene duplication, and are found in most vertebrates. The epididymis is highly exposed to infections by pathogens due to its anatomical position close to the urethra, and the expression of several beta defensins are entirely restricted to the epididymis. Examples of beta defensins expressed in the epididymis are DEFB104A, DEFB106B, DEFB118, DEFB129, DEFB132 and DEFB136.
Proteins expressed by the epididymal glands related to sperm maturation and fertilization
The SPINK2 gene encodes a serine protease inhibitor important for protection against proteolytic degradation of different tissues. SPINK2 is a strong inhibitor of acrosin (ACR), a trypsin-like protein expressed in spermatozoa that is important during the sperm penetration of the egg. CRISP1 encodes a member of the cysteine-rich secretory protein family 1 and plays a key role in the process of fertilization. CRISP1 is secreted in the epididymis and binds to the post-acrosomal region of the sperm head where it plays a role in the fusion of the sperm and egg plasma membranes. The decapacitation of the sperm is a reversible reaction that inhibits the fertilizing ability of the sperm, which takes place in the epididymis. The capacitation process is executed in the female reproductive tract just before fertilization, and the gene BSPH1 encodes a protein involved in sperm capacitation. The gene sperm associated antigen 11B, or SPAG11B, encodes several isoforms that are important for sperm maturation. Some of these isoforms contain regions of similarity to the beta defensin family. As the beta defensins, the expression of this protein is androgen-dependent and this gene is also located near the defensin gene cluster on chromosome 8p23. SPAG11B is specifically expressed in the epididymal epithelium, on the cell surface and also secreted in the lumen. ADGRG2, encoding a G-protein coupled receptor, is another gene involved in male fertility. The epididymis-specific expression pattern of this protein is known from previous data and experiments, however, the function of this protein is yet to be elucidated.
Gene expression shared between the epididymis and other tissues
There are 73 group enriched genes expressed in epididymis. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, including epididymis, compared to all other tissues.
To illustrate the relation of epididymis tissue to other tissue types, a network plot was generated, displaying the number of genes with a shared expression between different tissue types.
Figure 2. An interactive network plot of the epididymis enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of epididymis enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to 3 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.
Epididymis shares most group enriched gene expression with testis. KIAA1210 is group enriched in testis, epididymis and seminal vesicle. The protein expression in testis is mainly detected in Sertoli cells, while the expression in the excretory male glands is restricted to the fibromuscular tissues surrounding the glandular epithelia.
The secretoglobin family 2A member 1, also called Mammaglobin B, is encoded by the gene SCGB2A1. Although the knowledge and function of this protein is limited, previous data have shown that SCGB2A1 binds androgens and other steroids. SCGB2A1 is highly homologous to Mammaglobin A, encoded by the SCGB2A2, but the tissue expression for these two related genes differs. SCGB2A2 is highly expressed in mammary glands, while SCGB2A1 is group enriched in the epididymis and cervical glands.
The epididymis also shares group enriched gene expression of several genes with non-reproductive organs. One example is the gene PKHD1 encoding a protein thought to be involved in tubulogenesis. Apart from the epididymis, our data shows that PKHD1 is group enriched at high levels in both kidney and pancreas.
The epididymis is a highly coiled, single tubular structure that connects each testicle to the vas deferens, and later the ejaculatory system. The epididymal tube measures around 6-8 meters and originates from the embryonic Wolffian duct (also called the mesonephric duct), as the other parts of the male excretory duct system. The main functions of the epididymis are to store, transport and protect the spermatozoa as they pass through the tube. The newly synthesized, immature sperm cells that enter the epididymis from the efferent ductules are unable to fertilize an oocyte until subjected to several biochemical modifications that occur in the highly dynamic luminal milieu of the epididymis. The epididymal fluid milieu, expressed by the specialized regions of the epididymis, the caput (head), corpus (body) and cauda (tail), is important for the sperm to acquire their forward motility during the transit through the epididymal glands. The epididymis participates in the uptake and processing of residual and apoptotic bodies, and debris from the process of spermatogenesis in the testis.
The epididymal duct can be divided into four segments; the initial segment, head, body and tail. The ducts are surrounded by concentric layers of fibromuscular tissue, rich in smooth muscle cells with blood vessels and nerves in the adjacent supporting stroma. The duct of the epididymis is lined with pseudostratified columnar epithelial cells called principal cells. The principal cells are the major cell type and they have straight non-motile stereocilia that enable extensive absorptive and secretory functions. Together with the peristaltic movement of the smooth muscle, the duct cells facilitate the flow of sperm through the epididymis to the vas deferens and connect excretory tubes. In addition to the principal cells, the epididymal duct also consists of several other types of epithelial cells called clear cells, narrow cells, basal cells, halo cells, and the mitochondria-rich apical cells. However, the functions of these cells are poorly understood. A few scattered macrophages and lymphocytes can also be present in the stromal compartment.
Here, the protein-coding genes expressed in epididymis are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize corresponding protein expression patterns of genes with elevated expression in epididymis.
Transcript profiling was based on a combination of two transcriptomics datasets (HPA and GTEx), corresponding to a total of 14590 samples from 50 different human normal tissue types. The final consensus normalized expression (nTPM) value for each tissue type was used for the classification of all genes according to the tissue-specific expression into two different categories, based on specificity or distribution.
Relevant links and publications
Uhlén M et al., Tissue-based map of the human proteome. Science (2015)