Multiplex tissue profiling - Testis

The main function of the testis is the production of sperm (spermatogenesis), which takes place in the seminiferous ducts. This complex process involves multiple cell types and cell states through several stages of differentiation and maturation. As part of the Tissue resource, we used multiplex immunohistochemistry-based fluorescence (mIHC/IF) for in-depth profiling of protein expression in germ cells and Sertoli cells. In the current version, 612 proteins have been analyzed in 13 cell types/cell states using 4 antibody panels.

  • 13 different cell types
  • 612 profiled proteins
  • 4 antibody panels


The testis panels

The antibody panels for testis were developed with the aim i) to capture the transition of spermatogonial stem cells to preleptotene spermatocytes (Spermatogonia panel), ii) to identify the expression of proteins during spermatocyte differentiation and meiosis (Spermatocytes panel), iii) to characterize the proteins during sperm transformation, a process called spermiogenesis (Spermatids panel), and iv) mapping out the proteins Sertoli-specific proteins (Sertoli cells panel). The mIHC/IF technique is based on the overlap between the location of the candidate protein and location of the marker proteins targeted by the panel antibodies. Figure 1 shows the different cell types/states/structures, and the corresponding marker proteins included in each panel. For more information about the multiplex panels, please visit Assays & annotations.


Figure 1. Schematic depictions of the four testis panels Spermatogonia (A), Spermatocytes (B), Spermatids (C) and Sertoli cells (D) on the left with a list of cell types, representing colors and marker proteins on the right. SPG = spermatogonia; SPC = spermatocyte; SPT = spermatid.


Protein expression in testis

Candidate proteins for in-depth profiling were selected from the Tissue resource based on cell type specific staining pattern using conventional immunohistochemistry. Multiplex immunohistochemistry has allowed us to analyze and visualize protein expression in testis at a higher resolution than ever before. antibody panels. For testis, protein expression was analyzed in spermatogonia, spermatocytes, spermatids, and Sertoli cells.

Table 1. The number of proteins mainly located to different cell types/subcellular locations in testis based on manual annotation.

Antibody panel
Main location
# proteins
Spermatogonia Spermatocytes 1 50
Spermatogonia Spermatogonia 0 134
Spermatogonia Spermatogonia 1 68
Spermatogonia Spermatogonia 2-3 81
Spermatogonia Spermatogonia 4 88
Spermatocytes Spermatids early 80
Spermatocytes Spermatids late 28
Spermatocytes Spermatocytes 1 98
Spermatocytes Spermatocytes 2 102
Spermatocytes Spermatocytes 3 201
Spermatids Spermatids early 1 36
Spermatids Spermatids early 1 & Spermatids early 2 28
Spermatids Spermatids early 2 16
Spermatids Spermatids late 1 29
Spermatids Spermatids late 2 63
Spermatids Spermatids late 2 & Spermatids late 3 45
Spermatids Spermatids late 3 51
Sertoli cells Sertoli cytoplasm 53
Sertoli cells Sertoli membrane 13
Sertoli cells Sertoli nuclei 44
Sertoli cells Spermatids 7
Sertoli cells Spermatogonia and spermatocytes 26

Protein expression in spermatogonia

In total, 235 proteins were profiled in four cell states of spermatogonia defined as spermatogonia 0, spermatogonia 1, spermatogonia 2-3 and spermatogonia 4. An additional marker protein for the first spermatocyte stage, here defined as spermatocyte 1, was included for seamless integration with the Spermatocyte panel. The spermatogonia antibody panel covers the development of spermatogonial stem cells to spermatocytes. Spermatogonia represent the initial phase of spermatogenesis, characterized by a diploid state and undergoing clonal expansion through mitosis before transitioning into preleptotene spermatocytes.

Spermatogonia 0

As shown in Table 1, 134 genes have main protein location in spermatogonia 0. An example of a protein located in spermatogonia 0 is MAGEB2, a cancer-testis antigen that may enhance the activity of RING finger E3 ubiquitin ligases. Other examples are SPOCD1, suggested to be involved in DNA methylation and silencing in male embryonic germ cells, and POU2F2, a transcription factor thought to regulate the gene expression of IL6 among other genes.


MAGEB2

SPOCD1

POU2F2

MAGEB2

SPOCD1

POU2F2

Spermatogonia 1

As shown in Table 1, 68 genes have main protein location in spermatogonia 1. An example of a protein located in spermatogonia 1 is HNF1B, a transcription factor involved in nephron and embryonic pancreas development and with no known function in testis. Another example is MS4A14 which is predicted to be a receptor involved in signal transduction.


HNF1B

MS4A14


HNF1B

MS4A14

Spermatogonia 2-3

As shown in Table 1, 81 genes have main protein location in spermatogonia 2-3. An example of a protein located in spermatogonia 2-3 is cyclin A2 (CCNA2) which regulates the transition through the G1/S and G2/M phases of the cell cycle. Two other examples are NANOS3 which maintains the undifferentiated spermatogonia population by regulating the cell cycle, and FBXO24 which is important for cellular protein homeostasis.


CCNA2

NANOS3

FBXO24

CCNA2

NANOS3

FBXO24

Spermatogonia 4

As shown in Table 1, 88 genes have main protein location in spermatogonia 4. An example of a protein located in spermatogonia 4 is EMG1 which is a conserved eukaryotic protein essential for the biogenesis of ribosomes. Other examples are TIMELESS interacting protein (TIPIN) which plays an important role in cell division by controlling DNA replication, and sarcoma antigen 1 (SAGE1), a testis-cancer antigen with unknown function.


EMG1

TIPIN

SAGE1

EMG1

TIPIN

SAGE1

Protein expression in spermatocytes

In total, 276 proteins were profiled in three cell states of spermatocytes defined as spermatocytes 1, spermatocytes 2, and spermatocytes 3. Two additional marker proteins for the early and late spermatids were included for seamless integration with the Spermatid panel. Spermatocytes cover the development period between spermatogonia and spermatids. During this stage of spermatogenesis, the cells undergo the first and second meiosis, after which, each stage 1 spermatocyte turns into a total of four early spermatids.

Spermatocytes 1

As shown in Table 1, 98 genes have main protein location in spermatocytes 1. Examples of proteins found in spermatocytes 1 are cyclin-dependent kinase 1 (CDK1) with a key role in the regulation of the cell cycle as well as RAF1, a critical regulatory link for many cell fate decisions like proliferation, differentiation, and survival. A last example is TTC23L which has an unknown function but is mainly found in testis according to immunohistochemistry and RNAseq.


CDK1

RAF1

TTC23L

CDK1

RAF1

TTC23L

Spermatocytes 2

As shown in Table 1, 102 genes have main protein location in spermatocytes 2. Solute carrier family 9 member A9 (SLC9A9) is an example of a protein found in spermatocytes 2; it counteracts organellar acidity by transporting ions across endosomal membranes. Other examples of proteins found in spermatocytes 2 are pleiotrophin (PTN) and PNMA5 both with no known literature related to the testis.


SLC9A9

PTN

PNMA5

SLC9A9

PTN

PNMA5

Spermatocytes 3

As shown in Table 1, 201 genes have main protein location in spermatocytes 1. An example of a protein found in spermatocytes 3 is geminin DNA replication inhibitor (GMNN) which is a critical regulator of the cell cycle. Other examples are BOLL, found to be important for G2/M transition, and centrin 1 (CETN1) with a role in the microtubule organizing center structure during the cell cycle.


GMNN

BOLL

CETN1

GMNN

BOLL

CETN1

Protein expression in spermatids

In total, 89 proteins were profiled in five cell states of spermatids defined as spermatids early 1, spermatids early 2, spermatids late 1, spermatids late 2 and spermatids late 3. The spermatid antibody panel covers the last stage in spermatogenesis, called spermiogenesis, where spermatids transform into mature spermatozoa. Spermatids are haploid cells derived from spermatocytes that undergo a series of events, including transformation from a round to an elongated shape, condensation of DNA, development of an acrosomal cap, and formation of a tail (flagella).

Spermatids early 1

As shown in Table 1, 36 genes have main protein location in spermatids early 1. An example of protein located in spermatids early 1 is HMGB2 which is a multifunctional protein involved in e.g. transcription regulation and immune response. Another example is SH3GLB1 which is suggested to regulate apoptotic signal pathways and mitochondrial membrane morphology.


HMGB2

SH3GLB1


HMGB2

SH3GLB1

Spermatids early 2

As shown in Table 1, 16 genes have main protein location in spermatids early 2. Examples of proteins located in spermatids early 2 is ARID3A, a transcription factor that may control the cell cycle, and SUN5 which is involved in the meiotic stage of spermatogenesis.


ARID3A

SUN5


ARID3A

SUN5

Spermatids late 1

As shown in Table 1, 29 genes have main protein location in spermatids late 1. An example of a protein located in spermatids late 1 is DDX20, a putative RNA helicase thought to be involved in spermatogenesis among several other processes. Another example is FAIM2 which regulates apoptosis in neuronal cells, and its role in testis remains to be elucidated.


DDX20

FAIM2


DDX20

FAIM2

Spermatids late 2

As shown in Table 1, 63 genes have main protein location in spermatids late 2. An example of a protein located in spermatids late 2 is calaxin (CLXN) which is suggested to regulate ciliary and flagellar motility. Another example is sperm acrosome associated 9 (SPACA9), a component of microtubules in spermatozoa flagella.


CLXN

SPACA9


CLXN

SPACA9

Spermatids late 3

As shown in Table 1, 51 genes have main protein location in spermatids late 3. An example of a protein located in spermatids 3 is SMCP, which is believed to be a mitochondria-associated protein important for sperm motility. Another example is LELP1, the function of which has yet to be determined.


SMCP

LELP1


SMCP

LELP1

Protein expression in Sertoli cells

In total, 102 proteins were profiled in three different subcellular locations in Sertoli cells called Sertoli cytoplasm, Sertoli membrane and Sertoli nuclei. An additional marker covering the spermatogonia and spermatocytes and one for spermatids is used. The Sertoli cells are crucial support cells for the germ cells in the seminiferous ducts providing structure, nutrients and isolating them from molecules in the blood that would otherwise disrupt the spermatogenesis. For this, they have a strongly folded cytoplasm enveloping the spermatogonia and have tight interactions with other Sertoli cells in the seminiferous ducts.

Sertoli cytoplasm

As shown in Table 1, 53 genes have main protein location in Sertoli cytoplasm. An example of a protein found in the Sertoli cytoplasm is CLEC4M with a function in cell adhesion and pathogen recognition. Two other examples are CARMIL1 and ABCD3 which have functions in regulating actin polymerization and fatty acid transport across peroxisome membranes respectively.


CLEC4M

CARMIL1

ABCD3

CLEC4M

CARMIL1

ABCD3


Sertoli membrane

As shown in Table 1, 13 genes have main protein location in Sertoli membrane. Sertoli membrane proteins are often involved in cell junctions like junctional cadherin 5 associated (JCAD) and claudin 11 (CLDN11). JCAD is not very well known except for its function in cell junctions whereas CLDN11 has been shown to have a major role in the formation of cell junctions by removing the intercellular space in a calcium-independent matter.


JCAD

CLDN11


JCAD

CLDN11

Sertoli nuclei

As shown in Table 1, 44 genes have main protein location in Sertoli nuclei. Sertoli nuclei staining proteins are often transcription factors like for example SRY-box transcription factor 9 (SOX9) which is a general expressed transcription factor in non-germ cells and WT1 which has an important role in cellular development and survival. A third example is the unknown C12orf56 which is found in the nucleolar portion of the Sertoli nuclei.


SOX9

WT1

C12orf56

SOX9

WT1

C12orf56


Relevant links and publications

Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
PubMed: 25613900 DOI: 10.1126/science.1260419

Karlsson M et al., A single-cell type transcriptomics map of human tissues. Sci Adv. (2021)
PubMed: 34321199 DOI: 10.1126/sciadv.abh2169

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

Lewis SM et al., Spatial omics and multiplexed imaging to explore cancer biology. Nat Methods. (2021)
PubMed: 34341583 DOI: 10.1038/s41592-021-01203-6

Tan WCC et al., Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Cancer Commun (Lond). (2020)
PubMed: 32301585 DOI: 10.1002/cac2.12023