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First published online 21 December 2006
doi: 10.1242/dev.02751

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Potency of testicular somatic environment to support spermatogenesis in XX/Sry transgenic male mice

Mayuko Ishii^1,*, Tsuyoshi Tachiwana^1,*, Anshin Hoshino^1,*, Naoki Tsunekawa¹, Ryuji Hiramatsu¹, Shogo Matoba¹, Masami Kanai-Azuma², Hayato Kawakami², Masamichi Kurohmaru¹ and Yoshiakira Kanai^1,

¹ Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Bunkyoku, Tokyo 113-8657, Japan.
² Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.

View larger version (173K): [in this window] [in a new window]   Fig. 1. Spermatogonial transplantation into testicular seminiferous tubules of XX/Sry and XY W/Wv (control) males. (A,B) HE staining of the intact XX/Sry and XY W/Wv testes at 8 weeks of age, showing lack of germ cells in most tubules of both testes. (C-J) The recipient testes were injected with spermatogonial-cell suspension prepared from immature ROSA26 (C-F), wild-type (G,H) and Sl/Sld (I,J) testes, and were then histologically examined at 3 months after transplantation. (C-F) LacZ staining of the transplanted testes, visualizing ROSA26-derived XY germ cells in XX/Sry (C,E) and XY W/Wv (D,F) testes. Asterisks indicate LacZ-negative Sertoli cells located at the basal region of the seminiferous tubules. Insets show higher-magnification images of boxed area. (G-J) HE staining of XX/Sry (G,I) and XY W/Wv (H,J) testes injected with the wild-type-derived (G,H) or Sl/Sld-derived (I,J) testicular cell suspension. Higher-magnification images of recipient tubules with donor germ cells are shown on the right. Scale bars: 50 µm in A,G,I and inset of E; 200 µm in E.

View larger version (132K): [in this window] [in a new window]   Fig. 2. Defective spermiogenesis of donor XY germ cells in recipient seminiferous tubules of XX/Sry males. (A-D) Anti-MVH (A,B) and anti-HSC70T (C,D) stainings of two serial sections of XX/Sry and XY W/Wv testes (brown staining). (C,D) Insets show higher-magnification images of HSC70T-positive spermatids: notice the abnormal morphology of all HSP70T-positive cells in XX/Sry tubules. (E-K) PAS staining (red, E-H) and transmission electron microscopic (I-K) analyses of transplanted XX/Sry and XY W/Wv testes, showing three main types of degenerating round spermatids (arrows) in recipient XX/Sry tubules: detached (E), piknotic (F,I) and large multi-nucleated giant cells (G,J). Arrowhead in G shows early elongated spermatids at steps 8-10. Asterisks indicate Sertoli cells in E-H; Sertoli cell process in I-K. (L) Quantitative analysis of transplanted XX/Sry and XY W/Wv testes showing the number of seminiferous tubules with round (steps 1-7; r-spt) or early (steps 8-10)/late (steps 11-16) elongated spermatids (e-spt) relative to the total number of tubules with spermatocytes (spc). The bar represents mean +/-standard error; asterisks indicate a statistically significant difference between the XX/Sry and XY W/Wv testes (**P<0.01). Scale bars: 50 µmin A-H; 2 µm in I-K.

View larger version (148K): [in this window] [in a new window]   Fig. 3. Transplantation of seminiferous tubule segments under testis capsules of XX/Sry and XY W/Wv males. (A-K) Segments of immature XY seminiferous tubules (2-3 weeks old, wild type) were transplanted under the testis capsules of recipient XX/Sry (A,C) and XY W/Wv (B,D) males. Tubule segments of the primary-recipient XX/Sry testes with EGFP-positive XY spermatogenic colonies (2.5 months after spermatogonial transplantation using Green mice as donors; E,F) were further transplanted under the testis capsules of the secondary XY W/Wv recipients (G-K). All grafts were then histologically examined at 4 weeks after transplantation. (A,B) PAS staining showing elongated spermatids inside transplanted XY seminiferous tubules (arrows) of both recipient testes. (C,D) Immunohistochemical staining with anti-HSC70T antibody visualizing normal elongated spermatids of donor seminiferous tubules transplanted in XX/Sry testes. Insets show higher-magnification images of the donor seminiferous tubules. (E,F) Fluorescent micrographs showing a primary XX/Sry recipient testis (E) and two isolated tubule segments (F) with EGFP-positive XY spermatogenic colonies. (G-K) Anti-HSC70T and anti-EGFP immunohistochemical staining (two serial sections in G,H; K) and PAS staining (I,J) showing the absence of normal elongated spermatids (broken arrow in G; H), and the presence of pyknotic (arrowhead in I,K; J) and abnormally large (arrow in I,K) spermatids of XY spermatogenic colonies inside the donor XX/Sry seminiferous tubules transplanted in the secondary XY W/Wv recipients testes. Scale bars: 50 µm in A-H; 10 µm in I-K.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Microarray and RT-PCR analyses of gene expression in isolated seminiferous tubules from XX/Sry and XY W/Wv testes. (A,B) Microarray expression analysis showing the top 20 genes that were (A) downregulated and (B) up-regulated in XX/Sry testes. The fold change represents the difference in the expression level in seminiferous tubules between XX/Sry and XY W/Wv testes; fold change in parentheses showing the difference in expression level in whole testis. (C) RT-PCR analysis of genes selected from the top-20 genes that were up- and down-regulated in XX/Sry seminiferous tubules.