Figure 1.
Structure and organisation of the human testis and seminiferous tubules.
A. Macroscopic appearance. The testis is divided into compartments (lobules) separated by connective tissue. There are an estimated 250 lobules per testis, which vary in size; for clarity only 7 lobules are illustrated here. B. (i) Diagrammatic representation of the highly convoluted organisation of 3 seminiferous tubules (blue, black, grey) within a lobule. (ii) A magnified cross section through the lobule in i reveals how the tubules would appear in thin microscopic sections. Dashed lines (above section) and solid lines (below section) join the contiguous tubules. C. Cross section through an individual seminiferous tubule. Spermatogonia, located at the basal lamina of the seminiferous tubules, comprise a heterogeneous population of diploid germ cells. These can be classified according to their morphologies and correspond to three main maturation stages: Adark spermatogonia, which are considered to represent the reserve stem cell population; highly proliferating Apale spermatogonia; and more mature B spermatogonia that give rise to primary spermatocytes. Primary spermatocytes undergo meiosis to form secondary spermatocytes that differentiate to form spermatids, which when fully formed are released into the central lumen as spermatozoa that progress to the rete testis. Spermatogenesis is supported by the presence of the somatic Sertoli cells. The vascular and connective tissue network lie external to the wall of the seminiferous tubule.
Table 1.
Testis samples used in this work.
Table 2.
Primary antibodies used for immunohistochemistry.
Table 3.
Immunohistochemical screening for microclones in testes of 6 men.
Figure 2.
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.
Figure 3.
Examples of microclones in samples 2–1 and 3–1.
A. Microclone 2–1_C40, located away from the basal lamina, is positive for MAGEA4 and OCT2 (single cell identified post hoc) but not for FGFR3. B. Microclone 3–1_C1, located within the lumen of the tubule is positive for MAGEA4, FGFR3 and OCT2 (2 cells identified post hoc). Panels above the set of images show the markers used to stain the sections and the analysis of the results, using the same scheme as in legend to Figure 2. Scale bars: 100 µm.
Figure 4.
Immunopositive tubules in sample 2–1.
A. Under low power magnification of section 05, several clusters of two or more tubules with strong MAGEA4 staining are visible. B. Higher power magnification of the boxed region in A. Five tubules with stronger MAGEA4 staining (*) are distinguishable from their neighbouring tubules with normal levels of MAGEA4 staining. In normal tubules, MAGEA4 stains the nucleus and cytoplasm of the spermatogonia on the basal lamina. In the immunopositive tubules additional MAGEA4-positive cells are present, forming a double row. C. Serial sections spanning 115 µm display consistently stronger staining for MAGEA4, FGFR3 and pAKT. Clusters of MAGEA4 positive cells are present in the lumen of this immunopositive tubule. No differences in staining for OCT2, Ki67, SAGE1 or SSX are observed. H&E staining (section 02) shows that the tubule in the centre contains few spermatocytes and no spermatids, whereas the tubule on the right hand side contains both spermatocytes and spermatids. A higher resolution image of the H&E staining is shown in Figure S4A. Scale bar: 100 µm.
Figure 5.
Examples of immunopositive tubules in samples 1–1, 2–1 and 3–1.
Immunopositive tubules in three testis samples identified by staining with MAGEA4 also show enhanced staining for FGFR3 and pAKT. There is no difference in intensity of OCT2 staining, although some of the additional cells in sample 3–1 are OCT2 positive. H&E staining reveals that spermatocytes and spermatids are present in samples 1–1 and 2–1, but in sample 3–1 only spermatogonia and Sertoli cells are present (higher resolution images of the H&E panels are shown in Figure S4B). Scale bars: 100 µm. Note that sections are not all shown in sequential order.
Figure 6.
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).
Figure 7.
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.