The proteins locally secreted in the male reproductive system
Our analysis of the human secretome shows 126 proteins that are locally secreted in the male reproductive system. These proteins are found in the testis or male excretory glands (epididymis, ductus deferens, prostate and seminal vesicles). The main function of the testis is the spermatogenesis, sperm production, and synthesis of male sex hormones. The vast majority of cells in the testis reside in the seminiferous duct, where the spermatogenesis occurs. Additional testis-specific cells are the hormone producing Leydig cells that are located outside the seminiferous ducts. The main function of the epididymis is to store sperm and to mature newly synthesized sperm before it is ejaculated. Several key proteins are secreted in the epididymis; these proteins will create a dynamic microenvironment in the seminal fluid to protect the spermatozoa, and to protect their passage through the ductus deferens during ejaculation. The prostate is a gland producing fluid that is one of the constituents in semen, together with fluid from the seminal vesicles and sperm from the testis. The prostate is composed of secretory glands and a specific smooth muscle rich fibromuscular stroma. The fluid produced by the prostate is a protective and nourishing vehicle for sperm cells. Prostatic secretion consists of lipids, proteolytic enzymes, acid phosphatase, fibrinolysin and citric acids.
Functions of proteins locally secreted in the male reproductive system
All proteins that are secreted in male reproductive system were classified according to function based on Uniprot molecular function and biological processes keywords. The annotations were prioritized in the following hierarchy: Blood coagulation, Complement pathway, Acute phase, Cytokine, Hormone, Neuropeptide, Growth factor, Receptor, Lectin, Transport, Developmental protein, Defence, Enzyme, Enzyme inhibitor, Transcription, Immunity, Cell adhesion. Each gene was assigned one function. The result of the analysis is represented in Figure 1. Several protein classes can be found in the male reproductive system. Our analysis shows that 44 genes encode proteins lacking annotated function while 17 of the known proteins are related to enzymatic functions and 14 proteins are involved in enzymatic inhibitory functions. Enzymes are often androgen regulated and involved in sperm maturation and protection, as well as fatty acid and cholesterol regulation in male reproductive fluids. 3 proteins were found to be involved in transportation, mostly binding small hydrophobic ligands for transportation in between cells. 4 proteins were found to be involved in the development and gonadal differentiation Other secreted proteins are binding proteins, serine proteases among others (n=9).
Tissue specificity and tissue distribution classification
The genes encoding proteins locally secreted in the male reproductive system were further analyzed with regard to mRNA expression and categorized according to tissue specificity and tissue distribution. A vast majority of genes showed either tissue enriched (n=108) or group enriched (n=8) mRNA expression, i.e. either at least four-fold higher mRNA level in one tissue or in a group of two to five tissues compared to all other tissues (Figure 2). A small subset of genes (n=7) were tissue enhanced, i.e. showing four-fold higher average mRNA levels in one or more tissues compared to the mean mRNA level. Moreover, for most genes, mRNA was detected either in a single tissue or in a few tissues (less than 30 percent of the analyzed tissues) (Figure 3).Figure 2. Number of genes encoding proteins that are locally secreted in male reproductive system, categorized according to tissue specificity. Categories include: tissue enriched, defined as mRNA level in one tissue at least four-fold higher than in all other tissues; group enriched, defined as four-fold higher average mRNA level in a group of two to five tissues compared to all other tissues; tissue enhanced, defined as four-fold higher average mRNA level in one or more tissues compared to the mean mRNA level of all tissues; expressed in all, defined as ≥ 1 nTPM in all tissues; and not detected, defined as < 1 nTPM in all tissues. Figure 3. Number of genes encoding proteins that are locally secreted in male reproductive system, categorized according to tissue distribution. Categories include: detected in all, defined as n=100%; detected in many, defined as 31%=< n <100%; detected in some, defined as 1< n <31%; detected in single defined as single n=1; and not detected, n=0.
Origin of proteins locally secreted in male reproductive system
The analysis of gene expression showed that most proteins annotated as locally secreted in the male reproductive system are produced in the epididymis (n=61) (Figure 4). Remaining proteins were encoded by genes enriched in either testis (n=39), prostate (n=4) or seminal vesicle (n=3).
Exampes of secreted proteins in male reproductive system
In the epididymis, the defensin family of secreted proteins is very important. Many of these proteins are produced by immune cells and several other epithelial cells throughout the human body, however, a few of them are only expressed in the epididymis. The epididymis is highly exposed to infections because of its anatomical location. The beta defensin works, as the name implies, as the first line of defense against pathogens, and these proteins have antimicrobial and antiviral functions. A couple of secreted beta defensins that are tissue enriched in the epididymis are DEFB118, DEFB106A and DEFB129. These are only a few examples of secreted proteins, however, our analysis found that there are 61 locally secreted proteins in the epididymis.
The main function of the testis is the spermatogenesis (sperm production). Our analysis found 39 secreted proteins in the testis. Several of the proteins are important in the merge of the sperm and egg, one of them is ACR, Acrosin. This is a proteinase with trypsin-like specificity present in the acrosome of mature spermatozoa where it is stored in the precursor form as proacrosin. The function of acrosin is the breakdown of the zona pellucida for allowing the sperm to reach the glycoprotein layer of the oocyte. Another interesting protein family is zona pellucida binding protein (ZPBP). ZPBP is one of several proteins thought to be involved in a secondary binding between the reactive acrosome and the extracellular matrix in the oocyte. Some proteins that are enriched in testis lack annotated function but belong to an important protein family. One such example of a largely unknown protein enriched in testis is the CRISP2. However, in epididymis we can find CRISP1 which is an indication that they possess similar functions. In epididymis, CRISP1 is secreted to the epididymal lumen where it binds to the post acrosomal region at the sperm head and plays a role in sperm to egg fusion.
The 4 prostate gland enriched secreted proteins found in our analysis of the secretome are part of the kallikrein protein family, KLK2, KLK3, KLK4 along with microseminoprotein beta (MSMB) which is a part of the immunoglobulin binding factor family. It is synthesized in the prostate glands and secreted in the seminal plasma with an inhibin-like activity. The expression of MSMB is found to be decreased in prostate cancer. The kallikrein protein family is a subgroup of serine proteases and the KLK3 protein is present in seminal plasma. This protein, also known as PSA when found in plasma, is used as a clinical biomarker for prostate cancer. KLK2 in the active enzymatic form works as a trypsin-like protease responsible for cleaving pro-prostate-specific antigen (pro-PSA). KLK4 is also believed to be carcinogenic, and in some tissues hormonally regulated.
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