An alternative interpretation of cellular ‘selfish spermatogonial selection’‐ clusters in the human testis indicates the need for 3‐D‐analyses

Abstract

SUMMARYThe 'selfish spermatogonial selection'-model was proposed to explain the paternal age effect (PAE) of some congenital disorders associated with point mutations in male germ cells. According to this, spermatogonia carrying pathogenic mutations gain a selection advantage over non-mutated spermatogonia which leads to an increased number of mutated spermatogonia and consequently spermatozoa over time. Recently, an immunohistochemical approach using the premeiotic marker melanoma antigen family A4 (MAGE A4) was undertaken by the Wilkie group to confirm the presence of microclones of putatively mutated spermatogonia in testes of elderly men. The objective of our study was the age-dependent assessment of testes from men with normal spermatogenesis using MAGE A4 immunohistochemistry to identify and corroborate cellular clusters indicative for 'selfish spermatogonial selection' in our cohort. We analyzed testicular tissues obtained from men with normal spermatogenesis assigned to three age groups [(1) 28.8 AE 2.7 years; (2) 48.1 AE 1 years; (3) 71.9 AE 6.8 years, n/group = 8]. We could detect very similar distribution patterns of MAGE A4-positive cells and the presence of several types of microclusters as reported previously. However, these cellular clusters, indicative for clonal expansion, were not only present in testes from elderly men but also in those from age group 1 and 2. Using graphical three-dimensional modelling, we identified that cross-section directions e.g. longitudinal sections might provoke misleading interpretation of spermatogonial clusters, in particular when the tissue processing is limited. Thus, appropriate fixation and embedding is needed for reliable analysis of testicular sections. We therefore propose a more careful interpretation of such spermatogonial clusters and recommend a 3-D analysis to unequivocally determine 'selfish spermatogonial selection'-manifestations.

Cellular correlates of selfish spermatogonial selection

“…Recently Pohl et al . (2016) have contributed a Short Communication that has questioned the interpretation of our previous work. Briefly, when they examined Bouin's‐fixed testis biopsies from men of three different age groups (~29, ~48 and ~72 years), these authors found no change in the prevalence of cellular clusters, whereas an increase with age might be expected if they were caused by selfish selection.…”

“…(2016) did not appear to take full account of] between two immunohistochemical phenomena visible in testis sections: (i) putative ‘microclones’, defined as small clusters of MAGEA4‐expressing spermatogonial cells located at the periphery or within the lumen of tubular cross‐sections of seminiferous tubules, and (ii) ‘immunopositive tubules’, characterized by abnormal staining of the entire circumference of the tubular cross‐sections. The latter were readily visible at low magnification, often observed in groups, and displayed an overall darker staining when compared to their neighbours.…”

“…In our view, all the work presented by Pohl et al . (2016) concerns this ‘microclone’ (or, in their words, ‘microcluster’) appearance, the significance of which remains to be determined. We agree that artefactual explanations for microclusters are possible, especially when only MAGEA4 staining is employed, as was the case in Pohl et al .…”

“…We agree that artefactual explanations for microclusters are possible, especially when only MAGEA4 staining is employed, as was the case in Pohl et al . (2016) – note that in addition to spermatogonia, weaker MAGEA4 expression can also be detected in spermatocytes. In Lim et al .…”

“…Whatever the significance of these microclones/microclusters may be, in our opinion, the approach taken by Pohl et al . (2016) did not address the primary biological question concerning whether selfish clones can be identified.…”