PMC7461256_01
Histological evaluation. a Representative picture of the right testis, taken from the middle section. Two tubules are shown, the left one presenting only primary spermatocytes (lined arrowheads) and degenerating germ cells (asterisk), whereas the right tubule shows additionally a small group of elongated spermatids (circle). Gray lined arrowheads: spermatogonia. b Representative picture of the left testis, taken from the lower pole. Two tubules are shown, the left one presenting single primary spermatocytes (lined arrowheads) and spermatogonia (gray lined arrowheads) correctly aligned to the basal membrane, and the right tubule showing high numbers of spermatogonia/spermatogonia-like cells, dislocated and no longer restricted to the basal membrane (black arrowheads) with a single spermatocyte. Hematoxylin eosin (HE) staining, bar 50 mum
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Fig1
Fig. 1). In the right testis, single seminiferous tubules with qualitatively preserved, but quantitatively severely reduced spermatogenesis (hypospermatogenesis) was detected in three of four biopsies, accompanied with a focal tubular atrophy and arrest of spermatogenesis at the level of primary spermatocytes (representative picture shown in Fig. 1a). Score counts ranged from 0 (no tubules with elongated spermatids) to 0.1 (1% of tubules with at least single elongated spermatids) (Fig. 1a). Most interestingly, tubules showing a massive increase in numbers of spermatogonia-like cells were prevalent (representative picture shown in Fig. 1b).
Immunohistochemical analysis of germ and Sertoli cells. Pictures a, c, and e show intact spermatogenesis (NSP) as control, pictures b, d, and f show spermatogonial arrest (patient's left testis). a, b By using germ cell marker MAGEA4, we were able to differentiate Adark spermatogonia (black arrowheads) and Apale spermatogonia (black arrows) in NSP. In spermatogonial arrest, cells were MAGEA4-positive and therefore identified as spermatogonia. Sertoli cells (circles) and single megalospermatocytes (asterisk) were not stained. c, d Vimentin was present in Sertoli cell cytoplasm (ovals) in NSP and arrest. The upper tubule in D contains single spermatocytes and shows normal vimentin distribution. In the lower tubule, less intense vimentin staining in Sertoli cells were visible. e, f No specific cytokeratin 18 staining was detected in NSP and arrested spermatogenesis. Aminoethyl carbazole (AEC) detection, bar 50 mum, inset as negative control without first antibody
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Fig2
Fig. 2). Fig. 2). By using melanoma associated antigen 4 (MAGEA4) as a germ cell marker for type A and B spermatogonia, spermatogonia were detected in NSP lining up the basal membrane (Fig. 2a) and the abundant cells inside the peculiar seminiferous tubules of the patient's left testis could be identified as spermatogonia, too (Fig. 2b). Moreover, by applying distinct markers for Sertoli cells (i.e. vimentin, Fig. 2c / d, and cytokeratin 18 (CK18), Fig. 2e / f), we were able to show that Sertoli cells in both control and patient were normal in regard to intermediate filament presence and location. In those tubules with excessive numbers of spermatogonia, distribution of vimentin was less (Fig. 2d).
Immunohistochemical analysis of cell sex hormone receptors. Pictures a, c, and e show intact spermatogenesis (NSP) as control, pictures b, d, and f show spermatogonial arrest (patient's left testis). a, b In both, control and spermatogonial arrest, androgen receptor (AR) was expressed in Sertoli cells (circle), peritubular myoid cells (lined arrows) and Leydig cells (lined arrowheads). c, d In both specimen, estrogen receptor alpha (ERalpha) was expressed in primary spermatocytes (black arrowhead) and spermatogonia (black arrows). e, f In NSP and spermatogonial arrest, membrane-bound G-protein coupled estrogen receptor 1 (GPER1) was expressed in peritubular myoid cells (lined arrows). AEC detection, bar 50 mum (inset bar 25 mum), inset as negative control without first antibody
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Fig3
Fig. 3). Staining of sex hormone receptors, such as androgen receptor (AR, Fig. 3a / b), estrogen receptor alpha (Fig. 3c / d), and membrane-bound G-protein coupled estrogen receptor 1 (GPER1, Fig. 3e / f) was comparable in the control as well as in patient samples displaying spermatogonial arrest, including those tubules with abundance of spermatogonia. 
Immunohistochemical analysis of cell proliferation (a, b) and cell death (c, d). Pictures a and c show intact spermatogenesis (NSP) as control, pictures b and d show spermatogonial arrest (patient's left testis). a, b Proliferation was tested by PCNA immunostaining and revealed intensely stained spermatogonia (black arrows) and primary spermatocytes (black arrowheads). In the spermatogonial arrest tubules, PCNA staining was detected in more spermatogonia compared to NSP. c, d TUNEL staining revealed apoptotic germ cells in NSP (red arrows) and to a higher extend in tubules with abundant spermatogonia. AEC detection, bar 50 mum (inset bar 25 mum), inset as negative control without first antibody
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Fig4
Fig. 4). Fig. 4). By this, we were able to show that proliferation rate seems to be higher in tubules of the patient's left testis revealing abundance of spermatogonia (Fig. 4a / b) compared to control. DNA fragmentation as shown with TUNEL staining was also considerably higher in specimens of the patient's left testis compared to the control (Fig. 4c / d).
Immunohistochemical analysis of blood-testis barrier proteins. Pictures a, c, and e show intact spermatogenesis (NSP) as control, pictures b, d, and f show spermatogonial arrest (patient's left testis). a, b ZO-1 staining in NSP (a) revealed a clear cytoplasmic Sertoli cell staining. In contrast, in the sample showing spermatogonial arrest (b), ZO-1 staining was absent in a tubule showing spermatogonia alone (right tubule), but was present in an adjacent tubule with single remaining primary spermatocytes (lined arrowheads). c, d The same staining behavior was identified for claudin-11, where a regular staining was detectable in NSP (c) and in single tubules with remaining primary spermatocytes (lined arrowheads) (d, left tubule). But no staining was detected in tubules with spermatogonia only (d, right tubule). e, f A similar staining pattern was identified for CX43 staining in NSP (e) and in single tubules with remaining primary spermatocytes (lined arrowheads) (f, left tubule). No staining was detected in tubules with spermatogonia only (f, right tubule). AEC detection, bar 50 mum (inset bar 25 mum), inset as negative control without first antibody
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Fig5
Fig. 5). Fig. 5). Sertoli cell function and blood-testis barrier formation was analyzed by ZO-1, claudin 11 and connexin (CX) 43 immunohistochemistry in control (Fig. 5a, c, and e) and spermatogonial arrest in the patient's left testis (Fig. 5b, d, and f).