Sertoli–immature spermatids disengagement during testis regression in the armadillo

in Reproduction
Correspondence should be addressed to M S Merani or M Zuccotti or S Garagna; Email: mmerani@fmed.uba.ar or maurizio.zuccotti@unipv.it or silvia.garagna@unipv.it

*(V Merico and J P Luaces contributed equally to this work)

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In nature, mammalian seasonal breeders undergo spermatogenetic arrest during the non-breeding season. In the large hairy armadillo Chaetophractus villosus, testis regression initiates with immature post-meiotic germ cells sloughing into the tubule lumen and continues with the death of the remaining spermatocytes. At the end of the regression period, only spermatogonia and Sertoli cells persist in the seminiferous epithelium. It has been suggested that cell sloughing is determined by changes in the adhesion complexes between Sertoli cells and spermatids, which are mediated by low intra-testicular testosterone levels. By immunofluorescence and Western blotting we studied key proteins of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks that contribute to the complex Sertoli/spermatid adhesion system throughout the eight stages of the seminiferous epithelium cycle in the comparison between active and regressing testes. In active testis, B1-integrin, laminin G3, N-cadherin, B-catenin, P-B-catenin-Tyr654, FAK, P-FAK-Tyr397, SRC, P-SRC-Tyr416 proteins present a spermatogenetic cycle-dependent localisation pattern, unmaintained in regressing testes. In the latter, quantitative variations and changes in the phosphorylation state of protein FAK, SRC and B-catenin contribute to the disassembly of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks, thus promoting the massive release of immature spermatids.

 

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    Periodic acid-Schiff-hematoxylin (PAS, a–h) of cross-sections depicting the eight stages (annotated with Roman numeral) of the cycle of the seminiferous epithelium in active testes. The eight stages are defined by characteristic cell-specific associations, the progressive changes of spermatid nuclei shape and the acrosome formation (magenta staining) (Luaces et al. 2013). During spermiogenesis, spermatid nuclei acquire a characteristic spoon-like shape, very large in size and extremely thin (Cetica et al. 1998). The loosely packed chromatin (Luaces et al. 2013) within a large and flat nucleus results in very dim DAPI fluorescence (red pseudocolor). Confocal images of anti-B1-integrin (i–p) and anti-laminin G3 (q–x) immunofluorescence staining; nuclei are counterstained with DAPI (red pseudocolor) and merged with the corresponding immunofluorescence images (ii–pi) and (qi–xi). The signal is present, as a small rod, at stage III round spermatid heads (k–ki and s–si, respectively; arrowheads). Then, it progressively extends and, at stage VIII, a thin thread outlines the anterior-lateral head region of elongating spermatids (p–pi, x–xi, arrows). The same pattern is maintained until when mature spermatids are released into the tubule lumen (stage V) (m–mi, u–ui, arrows). Stage I and II round spermatids were negative (i–ji; q–ri, arrowheads). The signal is also positive at metaphase spermatocytes (p–pi, x–xi, arrowheads). Spermatogonia, empty arrowheads. Spermatocytes, double arrowheads. round spermatids, arrowheads; elongating spermatids, arrows. Bar, 20 μm.

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    Confocal images of anti-FAK (a–h) and anti-P-FAK (i–p) immunofluorescence staining on cross-sections representative of the eight stages (annotated with Roman numeral) of the cycle of the seminiferous epithelium in active testes. Nuclei are counterstained with DAPI (red pseudocolor) and merged with the corresponding immunofluorescence images (ai–hi and ii–pi). FAK signals are present at the basal compartment throughout the eight stages of the seminiferous epithelium cycle. Post-meiotic cells are always negative (arrows and arrowheads). P-FAK-Tyr397 is temporally detected only at stages III–V (k–mi). Signals are visible at the basal compartment (empty arrowheads), at spermatocytes (double arrowheads) at stages IV and V and at round spermatids (m–mi, arrowheads) at stage V. The signals contour elongated spermatids (arrows) with higher intensity at stage V (m–mi). Cells at stages I–II (i–ji) and VI–VIII (n–pi) are negative to the immunoreaction.

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    Confocal images of anti-N-cadherin (green), anti-B-catenin (red) and anti-P-B-catenin-Tyr654 (white) immunofluorescence staining on active testis sections. Nuclei are counterstained with DAPI (white, aii–hii, or red, ii–pi, pseudocolors) and merged with the corresponding immunofluorescence images. N-cadherin and B-catenin signals always co-localise. At stages I (a–aii) and II (c–cii), immunofluorescent signals are detectable at spermatogonia (empty arrowhead), spermatocytes (double arrowheads) and elongating spermatids (arrows) whereas they are undetectable at round spermatids (arrowheads). At stage III (e–eii), the signals mark both the Sertoli cell stalks at round (arrowheads) and elongating (arrows) spermatids. The same pattern is present at stage IV (g–gii). At stage V (i–iii), the signal remains strong at the basal compartment, marking spermatogonia (empty arrowhead), spermatocytes (double arrowheads), round (arrowheads) and elongated spermatids (arrows). At stages VI (k–kii) and VII (m–mii), immunofluorescence is delineating both pre-leptotene (empty arrows, bottom left) and leptotene (empty arrows) spermatocytes. At stage VIII (o–oii), co-localised signals are present at MI and MII spermatocytes (double arrowheads). P-B-catenin-Tyr654. Round (b–ji, arrowheads), from stage I to stage V, and elongating spermatids (l–li) at stage VI are negative to the immunostaining. At stage VII, the head tips of step 7 elongating spermatids (n–ni, arrows) become brightly decorated. At stage VIII, a thread-like signal extends from the anterior tip of the spermatid heads (p–pi, arrows) and elongates towards the opposite pole (b–ji) until when, at stage V, elongated spermatids are ready to be released into the lumen. The stages of the cycle of the seminiferous epithelium are annotated with Roman numeral. Bar, 20 μm.

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    Confocal images of anti-SRC (a–h) and anti-P-SRC (i–p) immunofluorescence staining on active testis section. Nuclei are counterstained with DAPI (red pseudocolor) and merged with the corresponding immunofluorescence images (ai–hi and i–pi). From stage I (a–ai) to stage VIII (h–hi) diffused SRC immunofluorescence is visible from the basal compartment to the lumen, located in the Sertoli cells cytoplasm surrounding spermatocytes (double arrowhead), round (arrowheads) and elongating (arrows) spermatids. At stages V (e–ei) and VI (f–fi) the immunosignal stains spermatogonia (empty arrowheads); at stage VII leptotene (g–gi, empty arrowheads) and at stage VIII zygotene (h–hi, empty arrowhead) spermatocytes. P-SRC-Tyr416. Stages I–III (i–ki) and VI–VIII (n–pi) are negative to the immunostaining. At both stages IV (l–li) and V (m–mi), fluorescence is localised at the adluminal compartment, surrounding the head of elongated spermatids (arrows) and protruding towards the lumen of the tubule with a rod-like shape. The stages of the cycle of the seminiferous epithelium are annotated with Roman numeral. Bar, 20 μm.

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    Testis section stained with Periodic acid-Schiff-hematoxylin (PAS, a–d). At the onset of the process, regression phase I, epithelium dysregulation and cell detachment determine alterations of the typical cell associations that impede the precise identification of each stage of the cycle (annotated with Roman numeral). For this reason, the eight stages were grouped in representative categories (a–c). At regression phase II (d), consistent cell sloughing made stage assignment impossible. Confocal images of anti-B-integrin (ei–hi) and anti-laminin G3 (ii–li) immunofluorescence staining on testis sections during regression phase I (a–c) and II (d). Nuclei are counterstained with DAPI (red pseudocolor) and merged with the corresponding immunofluorescence images (e–h and i–l). At both regression phase I and II, B1-integrin and laminin G3 confocal images show diffused signals from the basal to the adluminal compartment. Only elongated spermatid heads (arrows, squared nuclei) are still decorated with a thin fluorescence thread. Nuclei are counterstained with DAPI (red pseudocolor). Spermatocytes, double arrowheads; round spermatids, arrowheads; elongating spermatids, arrows. Bar, 20 μm.

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    Confocal images of anti-FAK (a–d) and anti-P-FAK (e–h) immunofluorescence staining of testis sections during regression phase I and II. Nuclei are counterstained with DAPI (red pseudocolor) and merged with the corresponding immunofluorescence images (ai–di and ei–hi). At regression phase I, FAK immunostaining is positive at the basal compartments marking spermatogonia (empty arrows). Diffused signals projects also towards spermatocytes (c–ci, double arrowheads) at stages VI–VIII. At regression phase II, very dim signals are present at the basal compartment (d–di). At regression phase I, dim P-FAK immunofluorescence is present at the basal (e–fi, empty arrowheads) and, more dispersed, at the adluminal (e–fi, double arrowheads) compartment at stages I–V; whereas very bright signals are visible at the tip of elongating spermatid heads (arrows, g–gi). P-FAK immunostaining is negative at regression phase II (h–hi). The stages of the seminiferous epithelium cycle are annotated with Roman numeral. Bar, 20 μm.

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    Confocal images of anti-N-cadherin (green), anti-B-catenin (red) and anti-P-B-catenin (white) immunofluorescence staining during regression phase I and II. Nuclei are counterstained with DAPI (white, aii–dii, or red, ei–hi, pseudocolors) and merged with the corresponding immunofluorescence images. At both regression phase I and II, N-cadherin and B-catenin signals always co-localise. At regression phase I with positive signals at spermatocytes (a–cii, double arrowheads), at elongating spermatids from stage I to stage V (a–bi, arrows) and at round spermatids at stages IV–V (b–bi, arrowheads). At regression phase II, N-cadherin and B-catenin co-localise at leptotene and pachytene spermatocytes (d–dii, empty arrows and double arrowheads, respectively) and at elongated spermatocytes (d–dii, arrows). Desquamated round spermatocytes (right to the dashed line) are also positive. Elongating spermatid heads are positive to the P-B-catenin immunosignals at both regression phase I and II (e–hi, arrows). At the latter regression stage, the positive spermatids are present in the tubule lumen (right to the dashed line). The stages of the seminiferous epithelium cycle are annotated with Roman numeral. Bar, 20 μm.

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    Confocal images of anti-SRC (a–d) and of anti-P-SRC (e–h) immunofluorescence staining of testis sections during regression phase I and II. Nuclei are counterstained with DAPI (red pseudocolor) and merged with the corresponding immunofluorescence images (ai–di and ei–hi). Both SRC and P-SRC contour spermatogonia (empty arrowheads), spermatocytes (double arrowheads), round (arrowheads) and elongating (arrow) spermatids. At regression phase II, P-SRC (h–hi) is very bright. The stages of the seminiferous epithelium cycle are annotated with Roman numeral. Bar, 20 μm.

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    (A) Box plot of relative protein expression in active (A) or regressing (R) testes. Each box comprises 50% of the data (including second and third quartiles) and is bisected by the median value. The error bar indicates the min and max values recorded. The non-parametric Mann–Whitney test (see ‘Results’ section for details) revealed, for each protein, statistically significant differences when comparing the expression levels between A and R testes. For each target protein, representative immunoblotting images are reported for both A and R testes. Actin was used as quantitative reference marker. (B) Fold-change expression of mean quantitative protein values in the comparison between A and R testes. Protein quantitation is based on five blots, from five different animals, for each protein analysed, with the exception of laminin G3 that was quantified on three blots from three different animals.

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    Schematic drawing of the histology of the seminiferous epithelium showing germ cells at different stages of their differentiation. In active testes the proteins of the A6B1 integrin/laminin G3 and N-Cadherin/N-Cadherin interlocks, which are part of the apical ectoplasmic specialisation are localised (red mark) at elongating and elongated spermatids. In regressing testes their quantity, localisation (red mark) and phosphorylation profile (purple mark) changes in correlation to cell desquamation.

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