PMC

The sizes (A) and weights (B) of Gpr56^−/− testes are significantly reduced compared with Gpr56^+/− or wild-type testes. This defect in testis development is unlikely due to hormonal deregulation, since the levels of FSH (C), LH (D), and testosterone (E) do not differ significantly between the Gpr56^+/− and Gpr56^−/− mice. *: p < 0.0001, Student’s t-test.

A. H&E staining of testis sections from 2-week old (n = 3) or 10-week old (n > 8) Gpr56^+/− and Gpr56^−/− mice. A disruption of seminiferous tubules (T) in Gpr56^−/− sections is apparent at both stages. Images of low magnifications are shown on the left, with some areas (red box) shown in high magnifications on the right.
B. The distribution of germ cells and Sertoli cells are abnormal in the Gpr56^−/− testes. Sections of testes from 10-week old Gpr56^+/− (n = 4) and Gpr56^−/− mice (n = 4) were stained with the anti-GCNA1 antibody to detect germ cells (arrows) and the anti-SOX9 antibody to detect Sertoli cells (arrows). Images of low magnifications are shown on the left, with some areas (red box) shown in high magnifications on the right. Depletion of germ cells and disorganized localization of Sertoli cells are seen in the disrupted areas of the Gpr56^−/− testes. Scale bar: 50 μm (A, B).

Genital ridges from Gpr56^+/− and Gpr56^−/− embryos (n = 3 for each genotype and stage) were stained with the anti-AMH antibody to visualize Sertoli cells during testis cord development. Whole genital ridges are shown on the left in low magnification, with an area close to the mesonephros (red box) shown in high magnification on the right. The boundaries between mesonephros and gonads are outlined by thin white lines. M: mesonephric side; C: coelomic side. Scale bar: 20 μm. The images reveal that testis cord development in Gpr56^−/− embryos is comparable to wild-type embryos at E12.5 and E13.5. However, at E14.5, E16.5, and E18.5, disruption in the formation of the testis cords on the mesonephric side (M) of Gpr56^−/− embryos is evident. In contrast, the cords on the coelomic side (C) remain intact.

A. The distribution patterns of Gpr56 mRNA in the genital ridges from E16.5 Gpr56^+/+ (n = 3) and Gpr56^−/− (n = 3) embryos were examined by in situ hybridization using Gpr56 antisense probe. The probe specifically detected Gpr56 mRNA in wild-type but not in testis cords from Gpr56^−/− mice. Gpr56^−/− gonads often appear larger than Gpr56^+/− gonads of the same stage. The reason is not known. M: mesonephros; C: coelomic epithelium.
B. The distribution pattern of GPR56 protein in the genital ridges from E14.5 Gpr56^+/+ (n = 4) and Gpr56^−/− (n = 4) embryos was examined by co-immunostaining with the anti-GPR56 antibody (red) and anti-AMH antibody (green), which labels Sertoli cells. Colocalization of GPR56 and AMH was observed in the wild-type but not in the Gpr56^−/− gonads. Images of whole genital ridges are shown in low magnification on the left, with an area in each gonad (red box) shown in high magnification on the right.
C. GPR56 was specifically detected in the Gpr56^+/− Sertoli cell preparations. Sertoli cells were isolated from two 4-week old Gpr56^+/− and Gpr56^−/− mice, lysed, and probed with the anti-GPR56 antibody and the anti-SOX9 antibody on a western blot. The level of GAPDH in each lysate was used as the loading control.

A. Gpr56^+/− and Gpr56^−/− embryonic gonads and adult testes (n = 4 for each stage and genotype) were stained with the anti-laminin 1 antibody. Images of low magnifications are shown on the left, with selected areas (red boxs) shown in high magnifications on the right. Laminin lines the testis cords (T) in Gpr56^+/− gonads and testes, but its localization is disrupted within the poorly organized regions of the Gpr56^−/− testes. The boundaries between mesonephros and gonads are outlined by thin white lines. M: mesonephric side; C: coelomic side. Scale bar: 20 μm.
B. To examine the relationship between basement membrane and Sertoli cells in the absence of GPR56, E16.5 Gpr56^−/− gonads were co-stained with anti-laminin 1 antibody (red) and anti-AMH antibody (green). Images of low magnifications are shown on the left, with selected areas (red boxs) shown in high magnifications on the right. Sertoli cells appear to remain attached to laminin in the absence of GPR56.
C. The anti-SMA antibody was used to label the peritubular myoid (PM) cells in Gpr56^+/− and Gpr56^−/− testes isolated from pups at P4 (n = 2 for each genotype). Images of low magnifications are shown on the left, with selected areas (red boxes) shown in high magnifications on the right. In wild-type tubules, the SMA-positive cells are tightly associated with the Sertoli cells. This is also observed in the intact Gpr56^−/− tubules. In the disrupted area of Gpr56^−/− testes (bottom panel), the PM cells continue to remain associated with the Sertoli cells. Black arrows: SMA-positive cells. Red arrowheads: Sertoli cells.