Yasuda T , Oda S , Li Z , Kimori Y , Kamei Y , Ishikawa T , Todo T , Mitani H .

	Figure 1. Testis–ova were differentiating spontaneously in the non-irradiated p53(−/−) testis. Representative images of the secondary sex characteristics of p53(−/−) medaka adults that are genetically male (b), showing typical male-type external appearance of wt male medaka (a) such as rougher edges of dorsal fins (arrows in a and b) and sharply long anal fins (arrowheads in a and b). H&E-stained sections of non-irradiated testes of wt and p53(−/−) fishes at 6 months old. (c) wt testis. (d) p53(−/−) testis. (e) Enlarged view of the boxed area in (c). (f) Enlarged view of the boxed area in (d) showing a small number of characteristic cells positioned in the cysts with type A or B spermatogonia, of which the nucleolus was strongly H&E-stained and the nucleus was faintly stained (arrows in f). sgA, type A spermatogonia; sgB, type B spermatogonia; sec, spermatocytes; st, spermatids; sp, spermatozoa. Scale bars represent 50 μm
	Figure 2. Histological changes of testis–ova in a p53(−/−) testis after 5 Gy γ-ray irradiation. Histological changes of testis–ova in a p53(−/−) testis during 28 days (1 month) after irradiation with 5 Gy of γ-rays. The arrowheads in c, d, f, g and h indicate the H&E-stained pyknotic cells, and arrows in c, d, g and h indicate the testis–ova. (a) H&E-stained section 1 day after irradiation. (b) Enlarged view of the boxed area in (a), showing that no apparent histological changes. (c) H&E-stained section 3 days after irradiation. (d) H&E-stained section 7 days after irradiation. H&E-stained pyknotic cells (arrowheads in d) that were positive in immunostaining against anticleaved caspase3 (arrowheads in f) in contrast to no positive signals in control p53(−/−) medaka (e), showing that these pyknotic cells are apoptotic. Testis–ova (arrows in d) increased extensively in the cysts of type A and early type B spermatogonia. (e) Immunostaining against anticleaved caspase3 in control p53(−/−) medaka testis. (g) H&E-stained section 14 days after irradiation. (h) Enlarged view of the boxed area in (g), showing that H&E-stained pyknotic cells (arrowheads in d, f, g and h) were observed continuously, and the larger size of testis–ova when compared with those 7 days after irradiation were observed (open arrows in g and h). (i) H&E-stained section of testis 28 days after irradiation. (j) Enlarged view of the boxed area in (i), showing that almost of all testis–ova had disappeared from the testis. Scale bars represent 50 μm
	Figure 3. Time course of the number of testis–ova in γ-ray-irradiated p53(−/−) testes. The number of testis–ova were counted on the histological section at the center of testes in γ-ray-irradiated testes of p53-deficient medaka at 1 day, 7 days, 14 days and 28 days after irradiation, and in a non-irradiated testis of both wt and p53-deficient medaka testes (n=3 for all time points). The error bars represent s.d.'s of means
	Figure 4. Electron microscopic observations of testis–ova in p53(−/−) mutants 7 days after irradiation with γ-rays (5 Gy). Morphological changes of testis–ova after γ-ray (5 Gy) irradiation. (a) Electron microscopic observation of wt spermatogonia. (c) Enlarged view of the boxed area in (a) showing the germinal dense body (nuage) in the cytoplasm (arrowhead with n in c), which is closely associated with large aggregations of mitochondria (arrows with m in c). The nucleolus of a spermatogonia is shown by an open arrowhead in c. (d) In the cytoplasm of p53(−/−) testis–ova, larger and more electron-dense nucleoli (e.g., open arrowhead in d) are present. It is noticeable that an extensive number of mitochondria and more electron-dense nuage are present (arrow with m and arrowhead with n in d) compared with those of wt spermatogonia (arrows with m and arrowhead with n in c). (b) The testis–ova in the irradiated testis increased synchronously (arrowheads in b) and they had a characteristic appearance of short and thick chromatin (arrows with c in b and e). (e) Apoptotic condensed nuclei were observed nearby testis–ova (arrowheads in e). (f) Some testis–ova have synaptonemal complexes in the nucleus (arrows in f). Chromatin (c), mitochondria (m), nuage (n). Scale bars represent 2 μm in c and d; 5 μm in a, b, e and f
	Figure 5. Histograms of type A spermatogonia and testis–ova in p53(−/−) testes 7 days and 14 days after irradiation. Areas of type A spermatogonia and testis–ova were segmented and extracted from the pictures of p53(−/−) testes after 7 days and 14 days after irradiation with γ-rays (1 and 5 Gy) by image processing procedures based on mathematical morphology. As type A spermatogonia and testis–ova were not able to be identified definitely by their size, we prepared the histograms for the cells including both type A spermatogonia and testis–ova, calculated by the mathematical morphology-based image processing method. (a) p53(−/−) testes after 7 days with γ-ray 1 Gy, (b) p53(−/−) testes after 14 days with γ-ray 1 Gy, (c) p53(−/−) testes after 7 days with γ-ray 5 Gy and (d) p53(−/−) testes after 14 days with γ-ray 5 Gy
	Figure 6. Histological changes of wt testes during 1 month after γ-ray (5 Gy) irradiation. (a) H&E-stained section of wt testis 1 day after irradiation. Many pyknotic cells were observed in the cysts of early type B spermatogonia (arrowheads in a). (c) Immunohistochemistry against anticleaved caspase3 showing the presence of immuno-positive cells in the cysts of early type B spermatogonia (arrowheads in c), where pyknotic cells (arrowheads in a) are positioned, in contrast to no positive signal shown in control wt medaka testis (b). (d) Histological H&E-stained section of wt testis 7 days after irradiation, and (e) enlarged view of the boxed area in (d), showing the histological appearance of hypertrophied Sertoli cells. (f) A schematic representation of hypertrophied Sertoli cells (pink) of (e). (g) Histological H&E-stained section of wt testis 14 days after the irradiation, and (h) enlarged view of the boxed area in (g), showing the hypertrophied Sertoli cells. (i) Histological H&E-stained section of wt testis 28 days after irradiation and (j) enlarged view of the boxed area in (i), showing histological appearance of the clear restoration of spermatogenesis. Scale bars represent 50 μm
	Figure 7. Histological changes of testis–ova in p53(−/−) testes during 1 month after γ-ray (1 Gy) irradiation. (a) H&E-stained section of p53(−/−) testis 1day after irradiation showing no apparent histological changes. (b) Histological H&E-stained section of p53(−/−) testis 7 days after irradiation and (c) enlarged view of the boxed area in B, showing pyknotic cells (arrowheads in c) and increased testis–ova (arrows in c). (d) Histological H&E-stained section of p53(−/−) testis 14 days after irradiation and (e) enlarged view of the boxed area in (d), showing pyknotic cells (arrowheads in e) and testis–ova (arrows in e) were present. (f) Histological H&E-stained section of p53(−/−) testis 28 days after irradiation showing that spermatogenesis was restored almost completely, and that the number of testis–ova had decreased. Scale bars represent 50 μm
	Figure 8. Gene expressions in p53(−/−) testes after γ-ray (5 Gy) irradiation. (a) Transcript of 42Sp50 were amplified both in non-irradiated and irradiated p53(−/−) testes with γ-rays (5 Gy), as well as in p53(−/−) and wt Hd-rR ovaries, while not amplified in non-irradiated and irradiated wt testes. (b) Expression of 42Sp50 in irradiated p53(−/−) testes was investigated on histological sections by ISH and as clearly shown in the enlarged view (c) of the boxed area in b, testis–ova in the p53(−/−)-irradiated testes were positive (blue-stained cells; arrows in c). (d) Transcript of Oct4 were amplified both in non-irradiated and irradiated p53(−/−) testes with γ-rays (5 Gy), as well as in p53(−/−) and wt Hd-rR ovaries, while not amplified in non-irradiated and irradiated wt testes. (e) Transcripts of vitellogenin were not amplified from male liver cDNA of wt and p53(−/−), without irradiation and with γ-ray (5 Gy) irradiation at 7days and 14 days. However, they were clearly amplified from female liver of both control wt and p53(−/−). Scale bars represent 50 μm

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