Male tissues

2718 genes are classified as testis elevated out of which 1004 genes are highlighted as testis enriched. Based on the expression in epididymis 685 genes are classified as elevated compared to other tissue types, out of which 115 genes are highlighted as tissue enriched in epididymis. RNA expression levels in ductus deferens resulted in 152 genes classified as elevated in ductus deferens compared to other tissue types, out of which 5 genes are highlighted as tissue enriched in ductus deferens. 117 genes are classified as penis elevated out of which 0 genes are highlighted as penis enriched. Based on the expression in male glands 1018 genes are classified as elevated compared to other tissue types, out of which 51 genes are highlighted as tissue enriched in male glands.

Histological image of the pig tissues used in the analysis can be found in the pig tissue dictionary.

Testis

The testis is essential for reproduction, responsible for producing sperm cells through spermatogenesis. Testis also produces hormones important for the development of male sex characteristics. The two major parts of the testis is the seminiferous ducts, where spermatogenesis takes place, and the Leydig cells located outside the seminiferous ducts producing hormones.

Gene expression in testis is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 2 summarizes the number of genes in respective category. In total, 16867 genes are detected above cut of (1NX) in pig testis. The tissue distribution category highlights 454 genes only detected in testis while 2718 genes are classified as testis elevated compared to other tissues. Table 1 shows the overlap for the testis elevated genes and tissue distribution category.

Figure 2, (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 (NX≥1) in testis as well as in all other tissues. The combination of the two categories is shown in table 1.

Table 1, Number of genes in the subdivided categories of elevated expression and tissue distribution in testis

Testis enriched expression

Cells found in the testis are cells not found elsewhere in the body, and therefore testis is the tissue type with the most uniquely expressed genes (expression only detected in testis and not detected above cut of in other tissues types). Among the genes classified as testis enriched there is a very good overlap to the corresponding human analysis of testis expression. TSSK6, PRSS37 and CRISP2 are all highlighted as testis enriched in pig as well as human.

Table 2, The 10 genes with the highest level of enriched expression in testis. "mRNA (tissue)" shows the transcript level in testis as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in testis and the tissue with second highest expression level.

Immunohistochemical labeling of testis, targeting TNP1 using HPA044387 and CYP19A1 using HPA051194.

A notable differences between species is that the pig testis includes a larger number of Leydig cells compared to human testis, therefore resulting in several genes expressed by Leydig cells classified as testis elevated in pig but not human, for example genes that in human are classified as adrenal gland enriched and low expression in testis shows higher expression in pig testis and therefore classified as group enriched, for example CYP11A1, FDX1 and STAR.

Epididymis

After the sperm production in testis, spermatozoa are stored and protected in the epididymis, where an interactive and dynamic microenvironment in the fluid enable further maturation of the sperm cells and function as a protected storage of the cells until their passage further to the ductus deferens.

Gene expression in epididymis is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 3 summarizes the number of genes in respective category. In total, 15311 genes are detected above cut of (1NX) in pig epididymis. The tissue distribution category highlights 39 genes only detected in epididymis while 685 genes are classified as epididymis elevated compared to other tissues. Table 3 shows the overlap for the epididymis elevated genes and tissue distribution category.

Figure 3, (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 (NX≥1) in epididymis as well as in all other tissues. The combination of the two categories is shown in table 3.

Table 3, Number of genes in the subdivided categories of elevated expression and tissue distribution in epididymis

Epididymis enriched expression

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, therefore many of the genes classified as enriched in epididymis are defensins, such as DEFB125, DEFB129 and DEFB128.

Table 4, The 10 genes with the highest level of enriched expression in epididymis. "mRNA (tissue)" shows the transcript level in epididymis as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in epididymis and the tissue with second highest expression level.

Ductus deferens

The main purpose of the ductus deferens is to transport sperm cells from the epididymis to the ampulla close to the seminal vesicle and out to the urethra. The ductus deferens consists of a tube lined with ciliated pseudo stratified columnar epithelium, surrounded by smooth muscle.

Gene expression in ductus deferens is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 4 summarizes the number of genes in respective category. In total, 15527 genes are detected above cut of (1NX) in pig ductus deferens. The tissue distribution category highlights 2 genes only detected in ductus deferens while 152 genes are classified as ductus deferens elevated compared to other tissues. Table 5 shows the overlap for the ductus deferens elevated genes and tissue distribution category.

Figure 4, (A) The distribution of all genes across the five categories based on transcript specificity in ductus deferens as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in ductus deferens as well as in all other tissues. The combination of the two categories is shown in table 5.

Table 5, Number of genes in the subdivided categories of elevated expression and tissue distribution in ductus deferens

Ductus deferens enriched expression

Table 6, The 5 genes classified as enriched in ductus deferens. "mRNA (tissue)" shows the transcript level in ductus deferens as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in ductus deferens and the tissue with second highest expression level.

Penis

The corkscrew-shaped penis of the pig was sampled at the middle of the penis body. Cross section of consecutive tissue can be found at the pig tissue dictionary. The penis tissue includes the urethra, lined with epithelia, no glandular tissue and is mainly composed by connective and erectile tissue.

Gene expression in penis is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 5 summarizes the number of genes in respective category. In total, 15066 genes are detected above cut of (1NX) in pig apenis. The tissue distribution category highlights 5 genes only detected in penis while 117 genes are classified as penis elevated compared to other tissues. Table 7 shows the overlap for the penis elevated genes and tissue distribution category.

Figure 5, (A) The distribution of all genes across the five categories based on transcript specificity in penis as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in penis as well as in all other tissues. The combination of the two categories is shown in table 7.

Table 7, Number of genes in the subdivided categories of elevated expression and tissue distribution in penis

In global expression analysis, penis cluster separate from the other male tissues and insterad show a global expression relation to ear cartilage, synovial tissue and other soft tissue rich tissue samples. Penis and ear cluster share the expression of elastin ELN, although much higher in the elastic cartilage of the ear, and the expresison in penis is one second place. Extracellular matrix related protines seem to play an important part in the expression profile of penis tissue, TGFB3 regulating extracellular matrix proteins is highly expressed in penis. The extracellular matrix protein NOV, is an example of gene with speces difference, in human NOV is higlh expressed in the adrenal gland and the pig adrenal gland show very low expression level while pig penis show high expression.

Other examples of genes highly expressed by penis are GREB1 shared with epididymis, prostate and female tissues and HOXD12 that is shared with male glands and urinary bladder.

Male glands

The tissues grouped as male glands represents the male accessory glands (seminal vesicle, prostate, bulbourethral gland and urethral glands) differ slightly between human and pig, both anatomically and histologically. The seminal vesicle is much larger in pig compared to human, the prostate is positioned a little differently. Pig has a large paired tubuloalveolar gland (bulbourethral gland) and urethral gland is surrounded by bulbourethral muscle. The glands are all secretory and the products both activate and nourish the spermatozoa in the semen. The pig accounts for the largest ejaculate volume among domestic animals, and the pig semen includes a large amout of gel.

The seminal fluild secreted from seminal vesicle function as a vehicle for the spermatozoa transport, while the secreted product of prostate formes a vaginal plug and also promotes movement of the spermatozoa. Bulbourethral gland produces mucus for lubrication and for clearing the urethra from urine, and finally The highest expression level among the four tissues was used as representative expression for male glands when comparing to other tissue types.

Gene expression in male glands is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 6 summarizes the number of genes in respective category. In total, 16395 genes are detected above cut of (1NX) in pig male glands. The tissue distribution category highlights 9 genes only detected in male glands while 1018 genes are classified as male glands elevated compared to other tissues. Table 9 shows the overlap for the male glands elevated genes and tissue distribution category.

Figure 6, (A) The distribution of all genes across the five categories based on transcript specificity in male glands as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in male glands as well as in all other tissues. The combination of the two categories is shown in table 9.

Table 9, Number of genes in the subdivided categories of elevated expression and tissue distribution in male glands

Male glands enriched expression

Since the main function of the male glands is to secrete proteins, there is no suprise that most genes classified as male gland enriched are secretd proteins, and also highly abundant. Many of the highly enriched genes are due to the high expression in seminal vesicle, for example SPMI, PSP-II and BSP1. prostate also accounts for several highly enriched genes, such as MSMB.

*Table 10, The 10 genes with the highest level of enriched expression in male glands. "mRNA (tissue)" shows the transcript level in male glands as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in male glands and the tissue with second highest expression level.

Expression comparison between the different male glands

The expression variation category enables further details related to expression heterogeneity within the tissues that include grouped samples. In this case, expression profile from the 4 male glands (seminal vesicle, prostate, bulbourethral gland and urethral gland) are compared and resulted in 3366 genes highlighted as variable.

An example of a protein with variable expression levels within the male glands is the PRR15, which is highly expressed in bulborethral, urathral and prostate while seminal vesicle show much lower expression.

Immunohistochemical detected of PRR15 in pig prostate and seminal vesicle using antibody HPA048121

Group enriched expression

The developmental origin and hormonal regulation of reproductive tissues can be observed by studyin the genes classified as group enriched. Several genes are shared among the male reproductive tissues, for example ABCB5 expressed by epididymis and ductus deferens, FOXR2specifically expressed by testis and epididymis, ADAM29 expressed by testis, epididymis and seminal vesicle. Several genes are shared between epididymis and ductus deferens, as well as epididymis and seminal vesicle. One eample of a protein shared between epididymis, ductus deferens and seminal veiscle is the transcription factor PAX2, important during development, which is also expressed in kidney and endometrium.

Immunohistochemical detection of PAX2 in pig epididymis, ductus deferens, seminal vesicle and endometrium using antibody HPA047704.

In order to illustrate the relation of male tissue to other tissue types, a network plot was generated, displaying the number of genes shared between different tissue types. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, compared to all other tissues.

Figure 7. An interactive network plot of the male tissue enriched and group enriched genes connected to their respective enriched tissues (grey circles). Black circles shows tissues representing the male reproductive tissues. Red nodes represent the number of tissue 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.