Limits...
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.

Show MeSH

Related in: MedlinePlus

Proposed model linking the immunohistochemical observations to clonal expansion of PAE mutations in seminiferous tubules.A. Tubule undergoing normal spermatogenesis: spermatogonia (grey) adjacent to basal lamina (blue line) proliferate and differentiate (arrows) to spermatozoa in the lumen. B. An activating PAE mutation arises in a single spermatogonial cell (red cell). C. Altered cellular signalling associated with the mutation confers a proliferative or survival advantage to the mutant cell, leading to clonal expansion of spermatogonia and relative enrichment of mutant sperm. The clonally expanded cells retain the immunohistochemical features of the originating cell (such as MAGEA4 and FGFR3) and may also have altered markers of signal activation (pAKT) - forming localised microclones (i) or expanding around the circumference and along the tubule (ii). Process (ii), compatible with the appearance of the immunopositive tubules identified in this study, would account for the distribution and number of mutant cells containing PAE mutations previously determined by DNA studies [20], [21], [22], [24]. D. In rare cases, additional mutational events may arise and lead to the formation of spermatocytic seminoma, possibly via an ISS intermediate state.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3412839&req=5

pone-0042382-g007: Proposed model linking the immunohistochemical observations to clonal expansion of PAE mutations in seminiferous tubules.A. Tubule undergoing normal spermatogenesis: spermatogonia (grey) adjacent to basal lamina (blue line) proliferate and differentiate (arrows) to spermatozoa in the lumen. B. An activating PAE mutation arises in a single spermatogonial cell (red cell). C. Altered cellular signalling associated with the mutation confers a proliferative or survival advantage to the mutant cell, leading to clonal expansion of spermatogonia and relative enrichment of mutant sperm. The clonally expanded cells retain the immunohistochemical features of the originating cell (such as MAGEA4 and FGFR3) and may also have altered markers of signal activation (pAKT) - forming localised microclones (i) or expanding around the circumference and along the tubule (ii). Process (ii), compatible with the appearance of the immunopositive tubules identified in this study, would account for the distribution and number of mutant cells containing PAE mutations previously determined by DNA studies [20], [21], [22], [24]. D. In rare cases, additional mutational events may arise and lead to the formation of spermatocytic seminoma, possibly via an ISS intermediate state.

Mentions: Although the appearance of a ‘double layer’ of spermatogonia has been observed by electron microscopy previously in testes of elderly men, this was assoociated with spermatogenesis arrest and was proposed to result from the failure of type A spermatogonia to differentiate [28]. In our study, H&E staining revealed the presence of spermatocytes and spermatids in immunopositive tubules of two of three testes, suggesting that these tubules were still capable of spermatogenesis (Figures 4C, 5, S4). The morphology of the immunopositive tubules was noticeably different from that described for ISS, in which the entire tubule, including the lumen, is replaced by round cells of varying sizes. However, the anatomical distribution of affected tubules within the tissue section is reminiscent of ISS and is consistent with the intratubular spread of a mutant cell population characterised by abnormal, enhanced signal activation (Figure 7). The likely clonal nature of the immunopositive tubules is supported by lineage tracing experiments of spermatogonial stem cells in the mouse testis, in which stochastically labelled cells expanded over time along the seminiferous tubules over distances up to 8.5 mm [44]. In the context of tumourigenesis, Lee et al. [19] observed that colonies resulting from transplantation of germ cells transfected with an activating Hras mutation also followed a tubular course. Moreover, immunohistochemical and H&E analysis of cross-sections of tubules populated by germ cells over-expressing activating Hras showed that these murine tubules were characterised by a double layer of spermatogonia, reminiscent of the phenomenon we describe in human testes [45].


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)

Proposed model linking the immunohistochemical observations to clonal expansion of PAE mutations in seminiferous tubules.A. Tubule undergoing normal spermatogenesis: spermatogonia (grey) adjacent to basal lamina (blue line) proliferate and differentiate (arrows) to spermatozoa in the lumen. B. An activating PAE mutation arises in a single spermatogonial cell (red cell). C. Altered cellular signalling associated with the mutation confers a proliferative or survival advantage to the mutant cell, leading to clonal expansion of spermatogonia and relative enrichment of mutant sperm. The clonally expanded cells retain the immunohistochemical features of the originating cell (such as MAGEA4 and FGFR3) and may also have altered markers of signal activation (pAKT) - forming localised microclones (i) or expanding around the circumference and along the tubule (ii). Process (ii), compatible with the appearance of the immunopositive tubules identified in this study, would account for the distribution and number of mutant cells containing PAE mutations previously determined by DNA studies [20], [21], [22], [24]. D. In rare cases, additional mutational events may arise and lead to the formation of spermatocytic seminoma, possibly via an ISS intermediate state.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0042382-g007: Proposed model linking the immunohistochemical observations to clonal expansion of PAE mutations in seminiferous tubules.A. Tubule undergoing normal spermatogenesis: spermatogonia (grey) adjacent to basal lamina (blue line) proliferate and differentiate (arrows) to spermatozoa in the lumen. B. An activating PAE mutation arises in a single spermatogonial cell (red cell). C. Altered cellular signalling associated with the mutation confers a proliferative or survival advantage to the mutant cell, leading to clonal expansion of spermatogonia and relative enrichment of mutant sperm. The clonally expanded cells retain the immunohistochemical features of the originating cell (such as MAGEA4 and FGFR3) and may also have altered markers of signal activation (pAKT) - forming localised microclones (i) or expanding around the circumference and along the tubule (ii). Process (ii), compatible with the appearance of the immunopositive tubules identified in this study, would account for the distribution and number of mutant cells containing PAE mutations previously determined by DNA studies [20], [21], [22], [24]. D. In rare cases, additional mutational events may arise and lead to the formation of spermatocytic seminoma, possibly via an ISS intermediate state.
Mentions: Although the appearance of a ‘double layer’ of spermatogonia has been observed by electron microscopy previously in testes of elderly men, this was assoociated with spermatogenesis arrest and was proposed to result from the failure of type A spermatogonia to differentiate [28]. In our study, H&E staining revealed the presence of spermatocytes and spermatids in immunopositive tubules of two of three testes, suggesting that these tubules were still capable of spermatogenesis (Figures 4C, 5, S4). The morphology of the immunopositive tubules was noticeably different from that described for ISS, in which the entire tubule, including the lumen, is replaced by round cells of varying sizes. However, the anatomical distribution of affected tubules within the tissue section is reminiscent of ISS and is consistent with the intratubular spread of a mutant cell population characterised by abnormal, enhanced signal activation (Figure 7). The likely clonal nature of the immunopositive tubules is supported by lineage tracing experiments of spermatogonial stem cells in the mouse testis, in which stochastically labelled cells expanded over time along the seminiferous tubules over distances up to 8.5 mm [44]. In the context of tumourigenesis, Lee et al. [19] observed that colonies resulting from transplantation of germ cells transfected with an activating Hras mutation also followed a tubular course. Moreover, immunohistochemical and H&E analysis of cross-sections of tubules populated by germ cells over-expressing activating Hras showed that these murine tubules were characterised by a double layer of spermatogonia, reminiscent of the phenomenon we describe in human testes [45].

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