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First published online 15 March 2005
doi: 10.1242/jcs.01738

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A novel Sry-downstream cellular event which preserves the readily available energy source of glycogen in mouse sex differentiation

¹ Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113-8657, Japan
³ Department of Global Agricultural Sciences, The University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113-8657, Japan
² Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan

View larger version (91K): [in a new window]   Fig. 4. A center-to-pole pattern of glycogen accumulation along anteroposterior (AP) axis of developing XY gonads and close association between glycogen-rich cells and germ cells during early phases of testis differentiation. (A) Sagittal sections of XY gonads isolated at 14 ts (upper) and 15 ts (lower) stages. PAS staining. Regions I-V are equally divided along the AP axis of the gonad. PAS-positive cells at 14 and 15 ts are located in regions II and III, respectively. Interestingly, they are frequently found in an area near germ cells even at 14 and 15 ts stages (right panel). Right panels show higher magnification images, indicated by the boxed area in the corresponding left panel. Asterisk, germ cell; ms, mesonephros. Bar, 50 µm. (B) Transmission electron micrographs showing a direct association between glycogen-positive cells and germ cells in XY gonad at 15 ts. Right panel shows a higher magnification image, indicated by the broken rectangle in left panel. Arrows indicate glycogen granules. ce, coelomic epithelial cells; G, germ cells; GR, glycogen-rich cells. Bar, 5 µm. (C) All consecutive sagittal sections were stained with PAS reaction, and then the total number of positive cells in each gonad was measured separately in regions I-V, which were equally divided along the AP axis of the gonad (broken lines in A). The vertical axis represents the PAS-positive cell number per gonad, whereas the horizontal axis represents regions I-V of the gonads. In each graph, bars of the same color show the cell number in each region of the same gonad (14 ts, five gonads; 15 and 16 ts, each four gonads). PAS-positive cells are positioned predominantly in the middle regions (regions II to IV) at 14 ts. This center-restricted distribution clearly expands into the anterior (region I) and posterior (region V) edges from 15 to 16 ts.

View larger version (158K): [in a new window]   Fig. 5. Glycogen accumulation occurs in XY gonads with severely reduced germ cells that are experimentally induced by busulfan treatment. Sagittal sections showing the effects of germ cell loss on PAS-staining pattern in XY gonads at 11.5 (18 ts; upper plates) and 12.5 (30 ts; lower plates) dpc. PAS staining. No germ cells are detected in these sections of busulfan-treated XY gonads shown in plates B and D. In XY gonads treated with busulfan, PAS-positive cells are properly aggregated into slender cord-like structures (B) similar to those in control XY gonads at the same stage (A), despite the drastic reduction in number of germ cells. In the testes at 12.5 dpc, the testicular cords are formed in the differentiated testes without germ cells, and Sertoli cells show positive PAS staining in both control (C) and busulfan-treated (D) testes. asterisk, germ cell; ms, mesonephros. Bar, 50 µm.

View larger version (114K): [in a new window]   Fig. 1. A sex-dimorphic distribution of the glycogen deposits in the gonads at 11.5 dpc (18 ts stage). (A-D) Transverse sections of 11.5-dpc XX (A,C) and XY (B,D) embryos. Periodic acid Schiff (PAS) staining (red staining). In both XY and XX embryos, several tissues including notochord (nc), skeletal muscle (sm) and the area close to dorsal aorta (da) show positive PAS staining. However, in developing genital ridges, PAS reactions are observed only in the gonadal region of XY, but not XX, embryos. In the XY gonad, PAS-positive cells are located close to germ cells (asterisks) (D), whereas no positive cells are detected in XX gonad (C). Plates C and D show higher magnification images, indicated by the broken rectangle in plates A and B, respectively. asterisk, germ cell; ce, coelomic epithelium; da; dorsal aorta; ms, mesonephros; nc, notochord; nt, neural tube; sm, skeletal muscle. Bar, 50 µm. (E,F) Transmission electron micrographs showing an accumulation of glycogen granules (arrows; a massive glycogen deposit is indicated by `Gly') in the cytoplasm of gonadal somatic cells closely associated with germ cells in the XY (F), but not the XX, gonad (E). G, germ cell; Gly, glycogen granule. Bar, 1 µm. (G,H) Two serial sections of the 11.5-dpc XY gonad were pretreated with (H) or without -amylase (G) before PAS staining. -amylase digestion results in a complete loss of PAS reaction in the XY gonad. The insets show higher magnification of the cells indicated by arrows. asterisk, germ cell; ms, mesonephros. Bar, 50 µm.

View larger version (104K): [in a new window]   Fig. 2. Timing of the onset of glycogen accumulation in developing XY gonads and its accumulation in the 11.5-dpc XX male gonad of sex-reversal Sry transgenic mice. (A) Temporal patterns of PAS reactions (red staining; transverse sections) and immunostaining against SOX9 protein (brown staining; sagittal sections) in the XX (left) and XY (middle and right) gonads during early phases of sex differentiation. Similar to the temporal pattern of SOX9 expression, PAS-positive cells are first detected in the XY gonad isolated at 14 ts (arrow for PAS; arrowheads for SOX9). These cells rapidly increased in number, and then aggregated into cord-like structures at 18 ts. No PAS-positive cells are detected in XX gonads at any stages examined. Embryos at approximately 11.0 and 11.5 dpc show 12 and 18 ts, respectively. (B) Transverse sections of the 11.5 dpc (18 ts) embryos of XX wildtype, XY wildtype and XX Sry trangenic embryos stained with PAS. PAS reactions are seen in XY wildtype gonads, as well as in the XX gonads of male embryos carrying the Sry transgene. ms, mesonephros. Bar, 50 µm.

View larger version (76K): [in a new window]   Fig. 3. PAS reaction and anti-SF-1/Ad4BP or anti-SOX9 staining of two consecutive mirror sections of 11.5-dpc XY gonads demonstrating that glycogen accumulation occurs in pre-Sertoli cell lineage. PAS reaction (red staining) and immunohistochemical staining (brown staining) with anti-SF-1/Ad4BP (a marker for precursor cells of both Sertoli and Leydig cells; A,B) or anti-SOX9 (pre-Sertoli cell marker; C,D) Ab were performed using two consecutive mirror sections of the same PFA-fixed specimens. For comparison, images of immunostained mirror sections are computationally reversed. Plates B and D are the merged images of PAS reaction (left) and immunoreaction (right; reversed images) in plates A and C, respectively. Plates B and D also include higher magnification images (right), indicated by the boxed area in the corresponding left plate. PAS-positive cells clearly overlap with gonadal somatic cells expressing SF-1/Ad4BP (arrows in B) or SOX9 (arrows in D) in the XY gonads. ms, mesonephros. Bar, 50 µm.

View larger version (176K): [in a new window]   Fig. 6. Genital ridge organ cultures showing the time course of testis-specific glycogen accumulation (A) and testis-specific induction even in serum withdrawal (B). (A) Sagittal sections of genital ridges cultured with 10% horse serum-DMEM for 6, 12, 24 and 48 hours and stained with PAS. In XY explants isolated at 12 ts, PAS-positive cells are detected after being cultured for 6 hours (arrow in XY 12ts), and then rapidly increased in the gonadal area near germ cells from 12 to 24 hours. In XY genital ridges isolated at 10 ts, PAS-positive cells are induced in the gonadal region at 12-24 hours (arrow in XY 10 ts). By contrast, no PAS-positive cells are detected in the gonadal region of XX genital ridges even after being cultured for 48 hours (XX 12 ts). (B) Sagittal sections of XY (left and middle) and XX (right) genital ridges (12 ts) after 48-hour culture in medium with or without 10% horse serum and stained with PAS. Despite severe growth defects in the genital ridges, PAS-positive cells are found to be induced in the gonadal area of the XY, but not XX, genital ridge, suggesting no appreciable effect of serum withdrawal on sex-dimorphic glycogen deposition in vitro. ms, mesonephros. Bar, 50 µm.

View larger version (119K): [in a new window]   Fig. 7. Testis-specific glycogen accumulation does not require adjacent mesonephros in vitro. Sagittal sections of genital ridges (gonad + mesonephros; A,B,D,E) and genital ridges without adjacent mesonephros (gonad alone; C,F) isolated at 14 ts. PAS staining. Plates show the genital ridge after being cultured for 0 (A,D) or 24 (B,C,E,F) hours. In the 24-hour cultures, glycogen accumulation is induced in XY explants (A,B), but not in XX explants (D,E), of genital ridges. Similar to the pattern of glycogen accumulation in genital ridge cultures, PAS-positive cells are observed in XY explants without mesonephros (C), whereas no PAS-positive cells are detected in XX explants without mesonephros (F). ms, mesonephros. Bar, 50 µm.

View larger version (124K): [in a new window]   Fig. 8. Inhibition of PI3K inhibitor (+LY), but not MEK inhibitor (+PD), on testis-specific glycogen accumulation in developing XY genital ridges in vitro. Sagittal sections of genital ridges (10-11 ts) cultured in the absence (A,D) or presence of MEK inhibitor (50 µM, PD98059; B,E) or PI3K inhibitor (15 µM; LY294002; C,F) with 10% horse serum-DMEM for 48 hours and stained with PAS. In XY explants, the addition of LY294002 to the medium drastically reduced glycogen accumulation in the gonadal area of XY explants (C). By contrast, the MEK inhibitor PD98059 did not exert any obvious effect on glycogen accumulation in XY explants (B). Moreover, no appreciable glycogen accumulation is observed in XX genital ridges treated with these inhibitors (D-F). ms, mesonephros. Bar, 50 µm.

View larger version (98K): [in a new window]   Fig. 9. Phosphorylation levels of AKT (a downstream effector of PI3K) in developing genital ridges in vitro and in vivo. (A) Sagittal sections of genital ridges (10-11 ts) cultured in the absence (cont) or presence of PI3K inhibitor (15 µM; LY294002; +LY) for 48 hours, and stained with anti-phospho-AKT. Positive signals for anti-phospho-AKT staining are detected in gonadal somatic cells located near germ cells, with a strong signal detected in the XY explant (XY cont) but only a weak signal in the XX explant (XX cont). Anti-phospho-AKT signals are clearly reduced in XY explants cultured in the presence of the PI3K inhibitor (XY + LY). (B) Immunoblot analyses of phosphorylation levels of AKT in XY and XX genital ridges (10-11 ts) cultured for 24 hours in the presence or absence of LY294002. In control XY explants, the AKT phosphorylation level is higher than that in XX control explants, and the level is clearly reduced by addition of LY294002 both in XY and XX explants. Repeat experiments were performed three times, and similar results were obtained each time. (C) Transverse sections of 11.5-dpc embryos (18 ts) and stained with anti-phospho-AKT. Anti-phospho-AKT reactions are found in the ventral region of neural tubes and dorsal root ganglion at similar levels in both sexes, but the reactions in the gonadal region are higher in XY than in XX embryos in vivo. These positive reactions in XY gonads are found in somatic cells near germ cells (asterisks). In plates A and C, right panels show higher magnification images, indicated by the boxed area in the corresponding left panel. asterisk, germ cell; drg, dorsal root ganglion; ms, mesonephros; nt, neural tube. Bar, 50 µm.

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