Hyperplastic SV structure in the AMH-Sox17 Tg male mouse (F0)

(A) Schematic diagram of the DNA construct containing Sox17 coding sequences driven by the human AMH promoter. (B, C) Gross morphology and hematoxylin and eosin (H&E)-stained sections of testes from infertile Tg #27 and its wildtype littermates. In the Tg testis, normal spermatogenesis is partially observed in the convoluted seminiferous tubules (ST), while an expanded SV-like region including decidual cell aggregates is evident in the proximal area near the rete testis (RT) (asterisks in C). (D) Immunostaining for SOX17 and E-cadherin (E-cad; a marker for both RT epithelia and Aundiffer spermatogonia), showing ST regions (left two panels) and SV regions (rightmost panel) of the testis sections. The AMH-Sox17 Tg testis exhibits ectopic high expression of SOX17 in almost all of the Sertoli cells in both ST and SV. Asterisks in (D) indicate protruded SOX17-positive SV Sertoli cells within the E-cad-positive RT lumen. RT, rete testis; SV, Sertoli valve. Scale bars: 1 mm (B), 200 μm (C), 50 μm (D).

Morphological characterization of the hyperplastic SV structure in a novel AMH-Sox17 Tg line

(A) Immunofluorescence staining for acetylated tubulin (red) and cyclin D1 (green) in the testes from Tg (#26) mice, showing expanded SV Sertoli cells. (B) Transmission electron microscopy (TEM) analysis of a Tg (#26) testis at the RT-SV boundary (red arrow), revealing abnormal features of SV Sertoli cells, such as protruded SV (pSV) structures extending into the RT lumen (upper left in B), multiple layers of cell processes containing microtubule bundles (blue asterisks), and open extracellular spaces in the basal region of the RT-SV boundary (red asterisk). Lower panels (B’–B’’’) are magnified views of the upper right panel in B. A vertical dashed line at the center marks the region hidden by the grid (grid spacing is 10 μm). BL, multilayered SV-typical basal lamina; RT, rete testis; SV, Sertoli valve. Scale bars: 50 μm (A), 10 μm (B), 2 μm (B’–B’’’).

Morphometric analysis of spermatogenic quality in control (Sox17f/f), cKO (Sf1-Cre;Sox17f/f), Tg (Tg; Sox17f/f), and cKO;Tg (Sf1-Cre;Sox17f/f; Tg) testes

(A) H&E-stained sections of the epididymis and testes from control, cKO, Tg, and cKO;Tg males. Two representative testis images from both fertile and infertile cKO;Tg males are also presented. The lower panels present higher magnification images of damaged seminiferous epithelia. (B) Evaluation of spermatogenesis in cross-sections of convoluted seminiferous tubules (STs). The evaluation score (maximum 10 points) was calculated as the sum of the seminiferous tubule structural integrity score (Group A; 7 points) and the most advanced germ cell type score (Group B; 3 points). The two graphs on the right show the evaluation scores for each genotype (left; mean ± SD; *p<0.05; **p<0.01; one-way ANOVA) and the percentage of seminiferous tubules containing the most advanced germ cells (right). Defective spermatogenesis observed in cKO testes was rescued in the cKO;Tg double mutant testes. Scale bars: 50 μm (top two rows); 10 μm (bottom row) in A.

Partial normalization of the expanded SV structure in AMH-Sox17 Tg testes by cKO;Tg double mutants

(A) Immunostaining for SOX9 and SOX17 in the RT–SV–ST region of proximal testes in control, Tg, and cKO;Tg mice. Ectopic SOX17 expression is observed in Sertoli cells of Tg testes, whereas SOX17 expression is completely absent in RT cells of the cKO;Tg double mutants. The expanded region of SOX17/SOX9 double-positive SV Sertoli cells (yellow) seen in Tg testes appears to be reduced in cKO;Tg double mutants. (B) Immunostaining for SOX9 (green) and acetylated tubulin (SV marker; red) or E-cadherin (RT marker; red) demonstrates the restoration of proper SV formation in the RT–SV region of the cKO;Tg double mutants. Additional panels show immunostaining for SOX17 (red)/SOX9 (green) and HSP70 (elongated spermatid marker; red)/SCP3 (spermatocyte marker; green) in ST regions (left panels). (C) Quantitaive data of the nuclear density of Sertoli cells in the RT-SV region (mean ± SD; *p<0.05; **p<0.01; one-way ANOVA). RT, rete testis; SV, Sertoli valve. Scale bars: 200 μm (top row) and 50 μm (bottom row) in A and B.

Enhanced Rspo1 and Wnt4 expression in the RT-SV boundary of the testes in AMH-Sox17 Tg mice

RNA in situ hybridization showing the Rspo1 and Wnt4 expression profiles of RT-SV boundary in four genotypes: control (Sox17f/f), cKO (Sf1-Cre;Sox17f/f), Tg (Tg; Sox17f/f), and cKO;Tg (Sf1-Cre;Sox17f/f; Tg) testes. RT, rete testis; SV, Sertoli valve. Scale bar, 50μm.

Schematic representation of valve structure size regulation by the AMH–Sox17 Tg mouse line

RT-specific Sox17 cKO mice exhibit deformed Sertoli valve (SV) structures, resulting in defective spermiogenesis and male infertility (Fig. 3; Uchida et al., 2022). Ectopic expression of SOX17 in Sertoli cells induces excessive SV formation in the terminal segments of the seminiferous tubule (ST), along with elevated Rspo1 expression in the rete testis (RT). Notably, this ectopic SOX17 expression partially rescues epididymal sperm production and restores fertility in cKO;Tg males. These Sox17 Tg mice, displaying SV hyperplasia and its functional rescue, provide valuable models for investigating paracrine signaling mechanisms within the RT-SV region.