The testis-specific proteome

The testis transcriptome

Transcriptome analysis of the testis 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 testis compared to other tissues. Elevated expression includes three subcategory types of elevated expression:

• Tissue enriched: At least four-fold higher mRNA level in testis compared to any other tissues.
• Group enriched: At least four-fold higher average mRNA level in a group of 2-5 tissues compared to any other tissue.
• Tissue enhanced: At least four-fold higher mRNA level in testis compared to the average level in all other tissues.

Distribution, on the other hand, visualizes how many genes have, or do not have, detectable levels (nTPM≥1) of transcribed mRNA molecules in the testis compared to other tissues. As evident in Table 1, all genes elevated in testis are categorized as:

• Detected in single: Detected in a single tissue
• Detected in some: Detected in more than one but less than one-third of tissues
• Detected in many: Detected in at least a third but not all tissues
• Detected in all: Detected in all tissues

Figure 1. (A) The distribution of all genes across the five categories based on transcript specificity in testis as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (nTPM≥1) in testis as well as in all other tissues.

As shown in Figure 1, 1968 genes show some level of elevated expression in the testis compared to other tissues. The three categories of genes with elevated expression in testis compared to other organs are shown in Table 1. In Table 2, the 12 genes with the highest enrichment in testis are defined.

Table 1. The number of genes in the subdivided categories of elevated expression in testis.

Table 2. The 12 genes with the highest level of enriched expression in testis. "Tissue distribution" describes the transcript detection (nTPM≥1) in testis as well as in all other tissues. "mRNA (tissue)" shows the transcript level in testis as nTPM values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in testis and the tissue with the second-highest expression level.

Protein expression of genes elevated in testis

In-depth analysis of the highly enriched genes in testis using antibody-based protein profiling allowed us to create a map of where these proteins are expressed with regards to the various cell types in seminiferous ducts corresponding to the different phases of the spermatogenesis including spermatogonia, spermatocytes, spermatids and mature sperms as well as Sertoli cells and Leydig cells.

A refined characterization of the protein expression in testis of 712 testis elevated genes was performed by taking eight cell types into consideration in the annotation, unlike the standard annotation which includes two cell types. The complete set of genes with in-depth annotation in the testis can be accessed here.

Proteins specifically expressed in spermatogonia and preleptotene spermatocytes

Spermatogonia are diploid cells that form the basal layer of the seminiferous duct and present the initial phase of the spermatogenesis. The spermatogonia undergo asymmetric cell division resulting in two subtypes, type A cells that have stem cell like properties and maintain the spermatogonia population, and type B spermatogonia will continue to evolve into preleptotene spermatocytes.

Examples of genes expressed in premeiotic spermatogonial cells include DMRT1, a transcription factor primarily expressed in the nuclei of spermatogonia, which plays a key role in male sex determination and differentiation by controlling testis development and male germ cell proliferation. PASD1 is a cancer-associated antigen and a nuclear suppressor of the biological clock. Another example of a cancer-associated antigen is SAGE1. The gene MAGEC1 encodes a member of the melanoma antigen gene (MAGE) family. ELAVL2 is a RNA-binding protein and is together with the uncharacterized open reading frame C3orf22 shown to be expressed mainly in the spermatogonia.

DMRT1
PASD1
SAGE1
MAGEC1
ELAVL2
C3orf22

Proteins specifically expressed in pachytene spermatocytes

Spermatocytes are derived from type B spermatogonia and can be subdivided into primary spermatocytes, that enter the first meiosis, and secondary spermatocytes that enter the second meiosis to produce haploid spermatids. Several of the testis-specific proteins localized to spermatocytes are involved in testicular differentiation, proliferation and meiosis. Pachytene spermatocytes are meiotic cells at the very end of prophase I. They are easily recognizable by being the only meiotic germ cells observed on testicular histology sections due to the long duration of meiotic prophase I.

Examples of proteins specifically expressed in the (secondary) pachytene spermatocytes include AURKA, a well-known cell cycle-regulated kinase important for microtubule stability during chromosome segregation. LY6K may play a role in cell growth and sperm migration, while BEND2 interacts with DNA during chromatin restructuring or transcription. The structural synaptonemal complex protein SYCP3 is involved in recombination and segregation of meiotic chromosomes, and HMMR protein is involved in cell motility. The meiotic recombination protein DMC1 is important for repairing double-strand DNA breaks during mitosis and meiosis.

AURKA
LY6K
BEND2
SYCP3
HMMR
DMC1

Proteins specifically expressed in round and elongated spermatids

Spermatids are derived from secondary spermatocytes and these cells are subdivided into early or round spermatids, that are transcriptionally active, and late or elongated spermatids that are transcriptionally inert. The testis enriched proteins with known function and predominant expression in spermatids are for example involved in the conversion of nucleosomal chromatin and sperm development and maturation.

Examples of proteins specifically expressed in spermatids include TNP1, which is involved in the conversion of nucleosomal chromatin to the compact, non-nucleosomal form found in the sperm nucleus, and ACTL7B, a gene encoding a putative protein of unknown function with incomplete evidence of existence limited to the transcript level. The formation of the acrosome, a testis-specific organelle required for sperm-egg interactions, is formed during the meiotic development of the late spermatid into mature spermatozoa. An example of a gene specifically expressed in the acrosome is ACRV1. This gene is expressed in the acrosomal vesicle during spermatogenesis and is associated with the acrosomal membranes and matrix of mature sperm. The ACRV1 protein may be involved in sperm-zona pellucida binding or penetration.

TNP1
ACRV1

Following the maturation of late stage spermatids, the testicular end stage of spermatogenesis results in the formation of sperm. Sperm consists of uniflagellar cells that are localized in the lumen of seminiferous ducts. Specific proteins localized to mature sperm are involved in sperm functions, such as sperm motility and male fertility. Examples of genes specifically expressed in sperm include PRM2, a protamine whose function is to substitute for histones in sperm and package sperm DNA into a highly condensed, stable and inactive complex. Other examples are GAPDHS, an enzyme that catalyzes an important energy-yielding step in carbohydrate metabolism during spermatogenesis, and AKAP4, suggested to be a major structural component of sperm fibrous sheath, which also plays a role in sperm motility.

PRM2
GAPDHS
AKAP4

Proteins specifically expressed in Sertoli and Leydig cells

The interactions between germinal cells, Sertoli cells and Leydig cells are essential, both for the development of testis and for the progress of spermatogenesis. Of the highly enriched genes in testis, only a few are specifically expressed in Sertoli cells or Leydig cells. One example of a gene expressed in Sertoli cells is SLCO6A1, currently with evidence only at transcript level. The INSL3 gene, specifically expressed in Leydig cells, is suggested to be involved in the development of the urogenital tract and involved in intra-abdominal testicular descent.

SLCO6A1
INSL3

Gene expression shared between testis and other tissues

There are 295 group enriched genes expressed in testis. 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 testis, compared to all other tissues.

To illustrate the relation of testis 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 testis enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of testis 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 4 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.

The network plot reveals that a lot of group-enriched gene expression is shared with fallopian tube (n=55) and also brain (n=126).

An analysis of the shared gene expression between the testis and fallopian tube shows enrichment for genes related to cilia structure and movement. One of the group enriched genes in testis and fallopian tube that is involved in cilia function and movement is DNAI1. This gene encodes a member of the dynein intermediate chain family and the encoded protein is part of the dynein complex in motile cilia. The uncharacterized leucine-rich repeat-containing protein LRRC46 is also expressed in testis and ciliated cells in the fallopian tube. SH3GL3 is an example of a gene with shared expression in the testis and brain, and this protein seems to be implicated in endocytosis.

DNAI1 - testis
DNAI1 - fallopian tube
DNAI1 - caudate

LRRC46 - testis
LRRC46 - fallopian tube

SH3GL3 - testis
SH3GL3 - cerebral cortex

Testis function

The main functions of the testis are spermatogenesis, which is the production of haploid germ cells essential for reproduction, and synthesis of androgens (mainly testosterone) necessary for e.g. development of male sex characteristics. These two functions occur in the two histologically different regions of the testis. Spermatogenesis takes place in seminiferous ducts, whereas the production of androgens occurs in Leydig cells that are interspersed between the seminiferous ducts. During spermatogenesis, cells of the seminiferous ducts undergo meiosis which contains several specific events such as reduction of the number of chromosomes, condensation of nucleus and removal of excess cytoplasm within the sperm. The sperm cells acquire unique morphological features and structures, such as flagellum and acrosome, that are necessary for sperm motility and fertilization of the egg, respectively.

Figure 3. A schematic figure representing the cross section of a seminiferous tubule illustrating the stepwise process of spermatogenesis. Initially, immature cells (spermatogonia), located close to the basal membrane, divide by several rounds of mitosis into primary and secondary spermatocytes, that further divide by meiosis to form spermatids. The final step of spermatogenesis takes place in the lumen of the seminiferous tubule where spermatids differentiate into haploid sperm cells.

Testis histology

The outermost part of the testis is surrounded by the tunica albuginea which is a fibrous layer of connective tissue. The testis is structured into lobules separated by fibrous septa where each lobule contains 1-4 seminiferous ducts. The seminiferous ducts make up the majority of the testicular tissue and each seminiferous duct consists of germ cells and Sertoli cells. Germ cells undergo mitosis and meiosis and mature into sperm in the process of spermatogenesis. Spermatogenesis involves several complex steps that can be morphologically determined and visualized in cross sections of seminiferous ducts. The different stages of spermatogenesis include undifferentiated spermatogonia in the basal compartment followed by more luminal mature spermatocytes and spermatids, and the end product of this process which is mature sperm. The Sertoli cells are hormone producing non-dividing columnar cells that are attached to the basement membrane and exhibit cytoplasmic extensions around the germinal cells. Sertoli cells have irregular nuclei with a typical prominent nucleolus. Interspersed between the seminiferous ducts are the hormone-producing Leydig cells together with blood vessels, lymphatics, nerves and inflammatory cells.

The histology of human testis including detailed images and information about the different cell types can be viewed in the Protein Atlas Histology Dictionary.

Background

Here, the protein-coding genes expressed in testis are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize corresponding protein expression patterns of genes with elevated expression in testis.

Transcript profiling was based on a combination of two transcriptomics datasets (HPA and GTEx), corresponding to a total of 14590 samples from 54 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)
PubMed: 25613900 DOI: 10.1126/science.1260419

Yu NY et al., Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium. Nucleic Acids Res. (2015)
PubMed: 26117540 DOI: 10.1093/nar/gkv608

Fagerberg L et al., Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics. (2014)
PubMed: 24309898 DOI: 10.1074/mcp.M113.035600

Djureinovic D et al., The human testis-specific proteome defined by transcriptomics and antibody-based profiling. Mol Hum Reprod. (2014)
PubMed: 24598113 DOI: 10.1093/molehr/gau018

Pineau C et al., Cell Type-Specific Expression of Testis Elevated Genes Based on Transcriptomics and Antibody-Based Proteomics. J Proteome Res. (2019)
PubMed: 31429579 DOI: 10.1021/acs.jproteome.9b00351

Histology dictionary - testis