The epididymis-specific proteome
The epididymis functions mainly as a reservoir for sperm and the place where the 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 secretion of several key proteins by the epididymal epithelial cells in orderto create a an interactive and dynamic microenvironment in the fluid where the spermatozoa can stay protected, and fully mature during their passage through the convoluted tube. The transcriptome analysis shows that 68% (n=13419) of all human proteins (n=19613) are expressed in the epididymis and 313 of these genes show an elevated expression in epididymis compared to other tissue types.
An analysis of genes with elevated expression in the epididymis shows that the corresponding proteins are mainly secreted and expressed by the glandular compartment of the tissue. The analysis also showed that the gene products elevated in epididymis are often associated in defense mechanisms and protection against microorganisms and other pathogens, but also genes implicated in the maturation process of the immotile sperm.
- 93 epididymis enriched genes
- Most elevated genes encode proteins involved in defence processes, protection against bacteria and sperm maturation
- 313 genes defined as elevated in the epididymis
- Most group enriched genes are shared with testis
Figure 1. The distribution of all genes across the five categories based on transcript abundance in epididymis as well as in all other tissues.
313 genes show some level of elevated expression in the epididymis compared to other tissues. The three categories of genes with elevated expression in epididymis compared to other organs are shown in Table 1. The function and cellular localization of known genes with tissue enriched expression in epididymis (n=93), are well in-line with the function of the epididymis.
In Table 2, the 12 genes with the highest level of expression among 93 enriched genes are defined.
Table 1. Number of genes in the subdivided categories of elevated expression in epididymis.
||Number of genes
||At least five-fold higher mRNA levels in a particular tissue as compared to all other tissues
||At least five-fold higher mRNA levels in a group of 2-7 tissues
||At least five-fold higher mRNA levels in a particular tissue as compared to average levels in all tissues
||Total number of elevated genes in epididymis
Table 2. The 12 genes with the highest level of enriched expression in epididymis. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. "mRNA (tissue)" shows the transcript level as TPM values, TS-score (Tissue Specificity score) corresponds to the score calculated as the fold change to the second highest tissue.
||defensin beta 105B
||defensin beta 106B
||defensin beta 128
||sperm associated antigen 11A
||defensin beta 118
||defensin beta 121
||defensin beta 105A
||sperm associated antigen 11B
||defensin beta 110
||binder of sperm protein homolog 1
||defensin beta 107B
||defensin beta 107A
Some of the proteins predicted to be membrane-spanning are intracellular, e.g. in the Golgi or mitochondrial membranes, and some of the proteins predicted to be secreted can potentially be retained in a compartment belonging to the secretory pathway, such as the ER, or remain attached to the outer surface of the cell membrane by a GPI anchor.
The epididymis transcriptome
An analysis of the expression levels of each gene made it possible to calculate the relative mRNA pool for each of the categories. The analysis shows that 73% of the mRNA molecules derived from epididymis correspond to housekeeping genes and only 20% of the mRNA pool corresponds to genes categorized to be either epididymis enriched, group enriched or, epididymis enhanced. Thus, most of the transcriptional activity in the epididymis relates to proteins with presumed housekeeping functions as they are found in all tissues and cells analyzed.
Gene Ontology-based analysis of all the 313 genes elevated in epididymis indicates a clear overrepresentation of proteins associated with defence processes and reactions against bacterial insult and other organisms. A vast majority of the 313 genes elevated in epididymis encodes secreted proteins.
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 are 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 the member of the cysteine-rich secretory protein family 1 and plays a key role in process of fertilization. CRISP1 is secreted in the epididymis. It binds to the post-acrosomal region of the sperm head where it plays a role in 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, and 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 the 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 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.
Genes shared between the epididymis and other tissues
There are 88 group enriched genes expressed in the epididymis. Group enriched genes are defined as genes showing a 5-fold higher average level of mRNA expression in a group of 2-7 tissues, including epididymis, compared to all other tissues.
In order to illustrate the relation of epididymis to other tissue types, a network plot was generated, displaying the number of genes shared 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 the largest number of genes 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 between 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 several genes with non-reproductive organs. One example is the gene SLC27A2 encoding an isozyme involved in the conversion of free long-chain fatty acids into fatty acyl-CoA esters. In the liver, SLC27A2 is believed to be implicated in bile acid metabolism. Apart from the liver, our data shows that SLC27A2 is group enriched at high levels in the epididymis and kidney as well.
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 is 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 occurs in the highly dynamic luminal milieu of the epididymis. The epididymal fluid milieu, expressed by the specialized regions of the epididymis — namely, the caput (head), corpus (body) and cauda (tail) - each region closely regulated by androgen factors and other steroids, 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 in 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 flow of sperm through the epididymis to the vas deferens and connecting 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.
The histology of human epididymis including detailed images and information about the different cell types can be viewed in the Protein Atlas Histology Dictionary.
Here, the protein-coding genes expressed in the epididymis are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize protein expression patterns of proteins that correspond to genes with elevated expression in the epididymis.
Transcript profiling and RNA-data analyses based on normal human tissues have been described previously (Fagerberg et al., 2013). Analyses of mRNA expression including over 99% of all human protein-coding genes was performed using deep RNA sequencing of 172 individual samples corresponding to 37 different human normal tissue types. RNA sequencing results of 1 fresh frozen tissues representing normal epididymis was compared to 171 other tissue samples corresponding to 36 tissue types, in order to determine genes with elevated expression in epididymis. A tissue-specific score, defined as the ratio between mRNA levels in epididymis compared to the mRNA levels in all other tissues, was used to divide the genes into different categories of expression.
These categories include: genes with elevated expression in epididymis, genes expressed in all tissues, genes with a mixed expression pattern, genes not expressed in epididymis, and genes not expressed in any tissue. Genes with elevated expression in epididymis were further sub-categorized as i) genes with enriched expression in epididymis, ii) genes with group enriched expression including epididymis and iii) genes with enhanced expression in epididymis.
Human tissue samples used for protein and mRNA expression analyses were collected and handled in accordance with Swedish laws and regulation and obtained from the Department of Pathology, Uppsala University Hospital, Uppsala, Sweden as part of the sample collection governed by the Uppsala Biobank. All human tissue samples used in the present study were anonymized in accordance with approval and advisory report from the Uppsala Ethical Review Board.
Relevant links and publications
Uhlén M et al, 2015. Tissue-based map of the human proteome. Science
PubMed: 25613900 DOI: 10.1126/science.1260419
Yu NY et al, 2015. Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium. Nucleic Acids Res.
PubMed: 26117540 DOI: 10.1093/nar/gkv608
Fagerberg L et al, 2014. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics.
PubMed: 24309898 DOI: 10.1074/mcp.M113.035600
Histology dictionary - the epididymis