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Selfish spermatogonial selection: evidence from an immunohistochemical screen in testes of elderly men.

Lim J, Maher GJ, Turner GD, Dudka-Ruszkowska W, Taylor S, Rajpert-De Meyts E, Goriely A, Wilkie AO - PLoS ONE (2012)

Bottom Line: The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia-caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively-represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men.We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance.However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT.

View Article: PubMed Central - PubMed

Affiliation: Clinical Genetics Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia-caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively-represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men. Previous analyses of DNA from randomly selected cadaveric testes showed that the levels of the corresponding FGFR2, FGFR3 and RET mutations exhibit very uneven spatial distributions, with localised hotspots surrounded by large mutation-negative areas. These studies imply that normal testes are mosaic for clusters of mutant cells: these clusters are predicted to have altered growth and signalling properties leading to their clonal expansion (selfish spermatogonial selection), but DNA extraction eliminates the possibility to study such processes at a tissue level. Using a panel of antibodies optimised for the detection of spermatocytic seminoma, a rare tumour of spermatogonial origin, we demonstrate that putative clonal events are frequent within normal testes of elderly men (mean age: 73.3 yrs) and can be classed into two broad categories. We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance. However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT. These findings support the concept that populations of spermatogonia in individual seminiferous tubules in the testes of older men are clonal mosaics with regard to their signalling properties and activation, thus fulfilling one of the specific predictions of selfish spermatogonial selection.

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Examples of putative microclones with different antigenic profiles in samples 1–1 and 1–2.A. MAGEA4 positive only (microclone no: 1–2_C1): nuclear immunoreactivity to MAGEA4 was identified independently (tagged with flag) in sections no.168, 171 and 175. This cellular cluster is negative for five additional markers as shown. Weaker, cytoplasmic MAGEA4 staining of primary spermatocytes is also present. B. MAGEA4, SSX, SAGE1, FGFR3 and Ki67 positive (microclone 1–1_C36). Additional FGFR3 positivity was determined post hoc. C. MAGEA4 and FGFR3 (microclone 1–1_C2): a large cluster of cells occupying the periphery of the tubule expresses MAGEA4 and FGFR3 on adjacent serial sections, but is negative for SSX, Ki67, SAGE1 and OCT2. Further screening was not possible because section 01 was the first section of the tissue block. D. SSX and SAGE1 (microclone 1–1_C41): one of the few examples where a microclone was negative for MAGEA4 expression. In this case, the cluster of cells is positive for SSX and SAGE1 only. The specificity of all markers including MAGEA4 is confirmed by their expression in spermatogonia situated at the periphery of the tubule (internal positive control). Scale bars: 100 µm. Tables above each figure display the antigenic profile (positive  =  coloured box with 1 (independent identification), or 0* (post hoc identification); negative  =  white box with 0) and length (pink bar) of the microclone. n.s: not stained. Cell counts for each positive section are also detailed.
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pone-0042382-g002: Examples of putative microclones with different antigenic profiles in samples 1–1 and 1–2.A. MAGEA4 positive only (microclone no: 1–2_C1): nuclear immunoreactivity to MAGEA4 was identified independently (tagged with flag) in sections no.168, 171 and 175. This cellular cluster is negative for five additional markers as shown. Weaker, cytoplasmic MAGEA4 staining of primary spermatocytes is also present. B. MAGEA4, SSX, SAGE1, FGFR3 and Ki67 positive (microclone 1–1_C36). Additional FGFR3 positivity was determined post hoc. C. MAGEA4 and FGFR3 (microclone 1–1_C2): a large cluster of cells occupying the periphery of the tubule expresses MAGEA4 and FGFR3 on adjacent serial sections, but is negative for SSX, Ki67, SAGE1 and OCT2. Further screening was not possible because section 01 was the first section of the tissue block. D. SSX and SAGE1 (microclone 1–1_C41): one of the few examples where a microclone was negative for MAGEA4 expression. In this case, the cluster of cells is positive for SSX and SAGE1 only. The specificity of all markers including MAGEA4 is confirmed by their expression in spermatogonia situated at the periphery of the tubule (internal positive control). Scale bars: 100 µm. Tables above each figure display the antigenic profile (positive  =  coloured box with 1 (independent identification), or 0* (post hoc identification); negative  =  white box with 0) and length (pink bar) of the microclone. n.s: not stained. Cell counts for each positive section are also detailed.

Mentions: Initially we developed and refined our immunohistochemical screen based on the analysis of two separate testis blocks from a 71 year old man (sample 1, Table 1). We flagged cellular clusters (see Materials and Methods and Figure S1 for definitions) by independently screening different sections with six antibodies (MAGEA4, FGFR3, Ki67, OCT2, SAGE1 and SSX); then, by comparing the positions of flagged cells on adjacent sections, we asked whether any of these flagged clusters showed physical contiguity between the sections. We found, indeed, that we were regularly able to identify such contiguous clusters of cells: we considered that the independent identification of such clusters in different sections made it less likely that these were simple staining artefacts, and more likely that they represented examples of groups of cells with localised alteration in antigen expression, compatible with their identification as putative microclones. In two separate blocks (samples 1–1 and 1–2) from one testis (of total thickness 215 µm), each comprising ∼3000 seminiferous tubule cross-sections, we identified 84 microclones; 62 (74%) were positive for MAGEA4 alone, 20 (24%) expressed MAGEA4 and other combinations of antigens, and 2 (2%) expressed SAGE1 and SSX only (Table 3). Selected examples of each of these antigen combinations are shown in Figure 2A–D, which also illustrates the varying patterns of size and cellular morphology that we encountered in microclones. A comprehensive illustration of the 84 microclones identified in samples 1–1 and 1–2 is presented in Dataset S2.


Selfish spermatogonial selection: evidence from an immunohistochemical screen in testes of elderly men.

Lim J, Maher GJ, Turner GD, Dudka-Ruszkowska W, Taylor S, Rajpert-De Meyts E, Goriely A, Wilkie AO - PLoS ONE (2012)

Examples of putative microclones with different antigenic profiles in samples 1–1 and 1–2.A. MAGEA4 positive only (microclone no: 1–2_C1): nuclear immunoreactivity to MAGEA4 was identified independently (tagged with flag) in sections no.168, 171 and 175. This cellular cluster is negative for five additional markers as shown. Weaker, cytoplasmic MAGEA4 staining of primary spermatocytes is also present. B. MAGEA4, SSX, SAGE1, FGFR3 and Ki67 positive (microclone 1–1_C36). Additional FGFR3 positivity was determined post hoc. C. MAGEA4 and FGFR3 (microclone 1–1_C2): a large cluster of cells occupying the periphery of the tubule expresses MAGEA4 and FGFR3 on adjacent serial sections, but is negative for SSX, Ki67, SAGE1 and OCT2. Further screening was not possible because section 01 was the first section of the tissue block. D. SSX and SAGE1 (microclone 1–1_C41): one of the few examples where a microclone was negative for MAGEA4 expression. In this case, the cluster of cells is positive for SSX and SAGE1 only. The specificity of all markers including MAGEA4 is confirmed by their expression in spermatogonia situated at the periphery of the tubule (internal positive control). Scale bars: 100 µm. Tables above each figure display the antigenic profile (positive  =  coloured box with 1 (independent identification), or 0* (post hoc identification); negative  =  white box with 0) and length (pink bar) of the microclone. n.s: not stained. Cell counts for each positive section are also detailed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412839&req=5

pone-0042382-g002: Examples of putative microclones with different antigenic profiles in samples 1–1 and 1–2.A. MAGEA4 positive only (microclone no: 1–2_C1): nuclear immunoreactivity to MAGEA4 was identified independently (tagged with flag) in sections no.168, 171 and 175. This cellular cluster is negative for five additional markers as shown. Weaker, cytoplasmic MAGEA4 staining of primary spermatocytes is also present. B. MAGEA4, SSX, SAGE1, FGFR3 and Ki67 positive (microclone 1–1_C36). Additional FGFR3 positivity was determined post hoc. C. MAGEA4 and FGFR3 (microclone 1–1_C2): a large cluster of cells occupying the periphery of the tubule expresses MAGEA4 and FGFR3 on adjacent serial sections, but is negative for SSX, Ki67, SAGE1 and OCT2. Further screening was not possible because section 01 was the first section of the tissue block. D. SSX and SAGE1 (microclone 1–1_C41): one of the few examples where a microclone was negative for MAGEA4 expression. In this case, the cluster of cells is positive for SSX and SAGE1 only. The specificity of all markers including MAGEA4 is confirmed by their expression in spermatogonia situated at the periphery of the tubule (internal positive control). Scale bars: 100 µm. Tables above each figure display the antigenic profile (positive  =  coloured box with 1 (independent identification), or 0* (post hoc identification); negative  =  white box with 0) and length (pink bar) of the microclone. n.s: not stained. Cell counts for each positive section are also detailed.
Mentions: Initially we developed and refined our immunohistochemical screen based on the analysis of two separate testis blocks from a 71 year old man (sample 1, Table 1). We flagged cellular clusters (see Materials and Methods and Figure S1 for definitions) by independently screening different sections with six antibodies (MAGEA4, FGFR3, Ki67, OCT2, SAGE1 and SSX); then, by comparing the positions of flagged cells on adjacent sections, we asked whether any of these flagged clusters showed physical contiguity between the sections. We found, indeed, that we were regularly able to identify such contiguous clusters of cells: we considered that the independent identification of such clusters in different sections made it less likely that these were simple staining artefacts, and more likely that they represented examples of groups of cells with localised alteration in antigen expression, compatible with their identification as putative microclones. In two separate blocks (samples 1–1 and 1–2) from one testis (of total thickness 215 µm), each comprising ∼3000 seminiferous tubule cross-sections, we identified 84 microclones; 62 (74%) were positive for MAGEA4 alone, 20 (24%) expressed MAGEA4 and other combinations of antigens, and 2 (2%) expressed SAGE1 and SSX only (Table 3). Selected examples of each of these antigen combinations are shown in Figure 2A–D, which also illustrates the varying patterns of size and cellular morphology that we encountered in microclones. A comprehensive illustration of the 84 microclones identified in samples 1–1 and 1–2 is presented in Dataset S2.

Bottom Line: The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia-caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively-represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men.We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance.However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT.

View Article: PubMed Central - PubMed

Affiliation: Clinical Genetics Group, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom.

ABSTRACT
The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia-caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively-represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men. Previous analyses of DNA from randomly selected cadaveric testes showed that the levels of the corresponding FGFR2, FGFR3 and RET mutations exhibit very uneven spatial distributions, with localised hotspots surrounded by large mutation-negative areas. These studies imply that normal testes are mosaic for clusters of mutant cells: these clusters are predicted to have altered growth and signalling properties leading to their clonal expansion (selfish spermatogonial selection), but DNA extraction eliminates the possibility to study such processes at a tissue level. Using a panel of antibodies optimised for the detection of spermatocytic seminoma, a rare tumour of spermatogonial origin, we demonstrate that putative clonal events are frequent within normal testes of elderly men (mean age: 73.3 yrs) and can be classed into two broad categories. We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance. However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT. These findings support the concept that populations of spermatogonia in individual seminiferous tubules in the testes of older men are clonal mosaics with regard to their signalling properties and activation, thus fulfilling one of the specific predictions of selfish spermatogonial selection.

Show MeSH
Related in: MedlinePlus