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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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3D reconstruction of immunopositive tubules in sample 1–1.A. (i) Five immunopositive tubular cross-sections (black arrows) in MAGEA4-stained section 51. The immunopositive tubules were followed by staining further sections at intervals of 4 or 6 slides (20 and 30 µm, respectively), until section 111. (ii). Although other immunopositive tubular cross-sections are present in section 111 (white arrows) and the intermediate sections, only those which could be traced back to section 51 (black arrows) were included in the reconstruction. Scale bars: 100 µm. B. 3D reconstruction of the immunopositive tubule. (i) The five immunopositive tubular cross-sections in section 51 were colour coded (green, pink, purple, blue and yellow arrows). The resolved 3D structure (with section 51 at top and section 111 at bottom) reveals that 4 of the 5 immunopositive cross-sections in section 51 (blue, purple, yellow and green) are part of the same tubule (joining where the colour margins blur together)(ii, iii, iv). Although they are in close proximity, it could not be demonstrated that the pink and green tubules are contiguous (iv). As the structure is highly convoluted, for clarity the scale of the z-axis has been increased 3-fold. A movie displaying the rotating 3D structure (shown to scale) is available as Video S1 (for description see Text S1).
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pone-0042382-g006: 3D reconstruction of immunopositive tubules in sample 1–1.A. (i) Five immunopositive tubular cross-sections (black arrows) in MAGEA4-stained section 51. The immunopositive tubules were followed by staining further sections at intervals of 4 or 6 slides (20 and 30 µm, respectively), until section 111. (ii). Although other immunopositive tubular cross-sections are present in section 111 (white arrows) and the intermediate sections, only those which could be traced back to section 51 (black arrows) were included in the reconstruction. Scale bars: 100 µm. B. 3D reconstruction of the immunopositive tubule. (i) The five immunopositive tubular cross-sections in section 51 were colour coded (green, pink, purple, blue and yellow arrows). The resolved 3D structure (with section 51 at top and section 111 at bottom) reveals that 4 of the 5 immunopositive cross-sections in section 51 (blue, purple, yellow and green) are part of the same tubule (joining where the colour margins blur together)(ii, iii, iv). Although they are in close proximity, it could not be demonstrated that the pink and green tubules are contiguous (iv). As the structure is highly convoluted, for clarity the scale of the z-axis has been increased 3-fold. A movie displaying the rotating 3D structure (shown to scale) is available as Video S1 (for description see Text S1).

Mentions: Whilst examining sections under low power magnification, we noticed that occasional tubules showed a distinct staining pattern in which the entire circumference exhibited enhanced immunopositivity for MAGEA4; moreover, several such tubules (which we term immunopositive tubules) often clustered in a localised part of the section and/or lay adjacent to each other (Figure 4 A, B). At higher power, we established that the increased MAGEA4 staining appeared attributable to the presence of additional immunopositive cells. These cells were mainly adjacent to the spermatogonia lying on the basal lamina, although in some cases additional cells were located higher in the germinal epithelium in a more lumenal position. In three of the testes examined (samples 1–1, 2–1 and 3–1) these immunopositive tubules were readily apparent (Figures 4, 5), but no such tubules were identified in the remainder (samples 4–1, 5–1, 6–1). By staining further sections with MAGEA4, we found that individual immunopositive tubules could be followed across all sections analysed in samples 2–1 and 3–1 (corresponding to block thicknesses of 115 and 110 µm, respectively) and up to 295 µm of tissue depth in sample 1–1. Despite the convoluted nature of the seminiferous epithelium, we could in some cases follow the U-turns taken by tubules using three-dimensional reconstruction; this analysis showed that neighbouring tubular cross-sections were part of the same unit and that these strong staining appearances were likely to encompass long contiguous segments of individual tubules, corresponding up to at least a millimetre of the tubular length (Figure 6, Text S1and Video S1; see also Figure S3).


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)

3D reconstruction of immunopositive tubules in sample 1–1.A. (i) Five immunopositive tubular cross-sections (black arrows) in MAGEA4-stained section 51. The immunopositive tubules were followed by staining further sections at intervals of 4 or 6 slides (20 and 30 µm, respectively), until section 111. (ii). Although other immunopositive tubular cross-sections are present in section 111 (white arrows) and the intermediate sections, only those which could be traced back to section 51 (black arrows) were included in the reconstruction. Scale bars: 100 µm. B. 3D reconstruction of the immunopositive tubule. (i) The five immunopositive tubular cross-sections in section 51 were colour coded (green, pink, purple, blue and yellow arrows). The resolved 3D structure (with section 51 at top and section 111 at bottom) reveals that 4 of the 5 immunopositive cross-sections in section 51 (blue, purple, yellow and green) are part of the same tubule (joining where the colour margins blur together)(ii, iii, iv). Although they are in close proximity, it could not be demonstrated that the pink and green tubules are contiguous (iv). As the structure is highly convoluted, for clarity the scale of the z-axis has been increased 3-fold. A movie displaying the rotating 3D structure (shown to scale) is available as Video S1 (for description see Text S1).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042382-g006: 3D reconstruction of immunopositive tubules in sample 1–1.A. (i) Five immunopositive tubular cross-sections (black arrows) in MAGEA4-stained section 51. The immunopositive tubules were followed by staining further sections at intervals of 4 or 6 slides (20 and 30 µm, respectively), until section 111. (ii). Although other immunopositive tubular cross-sections are present in section 111 (white arrows) and the intermediate sections, only those which could be traced back to section 51 (black arrows) were included in the reconstruction. Scale bars: 100 µm. B. 3D reconstruction of the immunopositive tubule. (i) The five immunopositive tubular cross-sections in section 51 were colour coded (green, pink, purple, blue and yellow arrows). The resolved 3D structure (with section 51 at top and section 111 at bottom) reveals that 4 of the 5 immunopositive cross-sections in section 51 (blue, purple, yellow and green) are part of the same tubule (joining where the colour margins blur together)(ii, iii, iv). Although they are in close proximity, it could not be demonstrated that the pink and green tubules are contiguous (iv). As the structure is highly convoluted, for clarity the scale of the z-axis has been increased 3-fold. A movie displaying the rotating 3D structure (shown to scale) is available as Video S1 (for description see Text S1).
Mentions: Whilst examining sections under low power magnification, we noticed that occasional tubules showed a distinct staining pattern in which the entire circumference exhibited enhanced immunopositivity for MAGEA4; moreover, several such tubules (which we term immunopositive tubules) often clustered in a localised part of the section and/or lay adjacent to each other (Figure 4 A, B). At higher power, we established that the increased MAGEA4 staining appeared attributable to the presence of additional immunopositive cells. These cells were mainly adjacent to the spermatogonia lying on the basal lamina, although in some cases additional cells were located higher in the germinal epithelium in a more lumenal position. In three of the testes examined (samples 1–1, 2–1 and 3–1) these immunopositive tubules were readily apparent (Figures 4, 5), but no such tubules were identified in the remainder (samples 4–1, 5–1, 6–1). By staining further sections with MAGEA4, we found that individual immunopositive tubules could be followed across all sections analysed in samples 2–1 and 3–1 (corresponding to block thicknesses of 115 and 110 µm, respectively) and up to 295 µm of tissue depth in sample 1–1. Despite the convoluted nature of the seminiferous epithelium, we could in some cases follow the U-turns taken by tubules using three-dimensional reconstruction; this analysis showed that neighbouring tubular cross-sections were part of the same unit and that these strong staining appearances were likely to encompass long contiguous segments of individual tubules, corresponding up to at least a millimetre of the tubular length (Figure 6, Text S1and Video S1; see also Figure S3).

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