Kawasaki T et al. (2006), Functional demonstration of the ability of a pr...

XB-ART-34361

Dev Growth Differ 2006 Oct 01;488:525-35. doi: 10.1111/j.1440-169X.2006.00890.x.

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Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis.

Kawasaki T , Imura F , Nakada A , Kubota H , Sakamaki K , Abe S , Takamune K .

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In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self-renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: fshb tle1 tle2

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	Fig. 1. Cultivation of testes obtained from juveniles that had just finished metamorphosis. (A, a) Section of testis of juvenile. (B, b, C, c) Sections of testes cultured for 4 weeks in the medium supplemented without (B and b) and with (C and c) 5 mU/mL of follicle stimulating hormone (FSH). (D, d) Section of testis from a juvenile 4 weeks of age after metamorphosis. Sections were stained with hematoxylin and eosin. Each higher magnification view of the testis (a, b, c or d) is an enlargement of each boxed area of the low magnification views (A, B, C or D). Note that by culturing a testis in the medium containing FSH, the spermatogonial cells differentiated into the primary spermatocytes. Scale bars represent 20 μm (a-d) and 100 μm (A-D), respectively. (E) Comparison of the number of spermatocysts consisting of primary spermatogonium (PG), early secondary spermatogonia (ESG), late-secondary spermatogonia (LSG), and primary spermatocytes (PC) between the testis cultured in the medium supplemented with 5 mU/mL of FSH () and the testis from juveniles which were reared after the metamorphosis for the same periods as cultivation (). To avoid an incorrect estimation caused by biased distribution of the spermatogenic cells in each section and by repeat counting of the same spermatocyst, we counted them in every eighth section. The vertical bar on each symbol represents the standard deviation of the mean in the examined testes, of which the number is shown with the symbols in the uppermost column.
	Fig. 2. Progression of spermatogenesis in testes by transplanting them under the abdominal skin of host juveniles. (A, a, B, b) Sections of host (A and a) and transplanted (B and b) testes obtained from a male frog. (C, c) Section of a transplanted testis obtained from a female frog. Eight weeks after the transplant, the testes were extirpated and their sections were stained with hematoxylin and eosin. Each higher magnification view of testis (a, b or c) is an enlargement of each boxed area of the low magnification views (A, B or C). Scale bars represent 30 μm (a-c) and 150 μm (A-C), respectively. ESG, early secondary spermatogonia; MS, mature sperm; PC, primary spermatocytes; PG, primary spermatogonium.
	Fig. 3. The number of spermatocysts in transplanted (T) and host (H) testes obtained from two males (Transplantation into Male, A and B) and in transplanted testes obtained from two females (T; Transplantation into Female, A and B). Immediately after the completion of metamorphosis, a pair of testes was obtained from the male, and one of them was fixed for examining the initial state of testis (IC) and the other was transplanted under the skin of another juvenile. Eight weeks after the surgery, testes were removed and fixed. To compare the state of those testes with that of normal testes, three testes were obtained from the frogs at 8 weeks of age after metamorphosis (No Surgery, A, B and C) and fixed. The number of the spermatocysts consisting of the spermatogenic cells at various developmental stages was estimated by counting them in each eighth section to avoid an incorrect estimation caused by biased distribution of the spermatogenic cells in each section and by repeat counting of the same spermatocyst. Letters at the bottom of the figure denote the stage of the spermatogenic cells examined: PG, primary spermatogonia; ESG, early secondary spermatogonia; LSG, late-secondary spermatogonia; PC, primary spermatocytes; Td, round spermatids; ELO, elongating spermatids; MT, mature sperm.
	Fig. 4. Progression of spermatogenesis in aggregates that were prepared by massing dissociated testicular cells obtained from juvenile testes. (A, a) Section of juvenile testis. (B) Dissociated testicular cells. (C, D, d) An aggregate prepared by massing the dissociated testicular cells in B (C) and its section (D and d). (E, e, F, f, G, g) Sections of aggregates. These had been transplanted under the abdominal skin of host juveniles and were extirpated and fixed 4 (E and e), 8 (F and f), and 20 (G and g) weeks after transplantation, respectively. Sections were stained with hematoxylin and eosin. Each higher magnification view of testis (a) or aggregates (d-g) is an enlargement of each boxed area of the low magnification views (A, D, E, F, G). Scale bars represent 20 μm (a, d, e, f, g), 100 μm (A, B, D, E, F, G) and 500 μm (C), respectively. PG, primary spermatogonium; ESG, early secondary spermatogonia; LSG, late-secondary spermatogonia; PC, primary spermatocytes; MS, mature sperm.
	Fig. 5. Tracing the destiny of a single primary spermatogonium obtained from transgenic juvenile testis. A genetically marked cell was mixed with non-marked testicular cells before making the aggregate that was transplanted subcutaneously. (A) Section of testis from transgenic juvenile. The section was stained with hematoxylin and eosin. (B) Dissociated testicular cells of transgenic juvenile. Arrow shows the largest cell in the cell population, which was picked up as primary spermatogonium. (C, D) Differential interference contrast microscopic image (C) and section (D) of a primary spermatogonium picked up from the dissociated testicular cell population in (B). The section was stained with hematoxylin and eosin. Note that the fixed specimen slightly shrank with the effect of fixative (D). (E, e, F, f, G, g) Fluorescent (E) and bright field (e) images, and a section (F, f, G, g) of aggregate that was extirpated from the host frog 8 weeks after transplantation. The section was stained with hematoxylin and eosin (G and g), followed by destaining the hematoxylin and eosin and successively staining with anti- GFP mouse monoclonal antibody and alkaline phosphataseconjugated antimouse IgG antibody (F and f). Each higher magnification view of aggregate (f or g) is an enlargement of each boxed area of the low magnification views (F and G). Scale bars represent 20 μm (A, C, D), 30 μm (f and g), 50 μm (B), 300 μm (F and G), and 500 μm (E and e), respectively. PG, primary spermatogonium; LSG, late-secondary spermatogonia; PC, primary spermatocytes.
	Fig. 6. Tracing the destiny of a single primary spermatogonium (PG) isolated from transgenic juvenile testis that had been cultured for 2 weeks in the medium supplemented with follicle stimulating hormone (FSH). A genetically marked PG was mixed with non-marked testicular cells before making the aggregate. Twelve weeks after subcutaneous transplantation of the aggregates, a fully grown aggregate (A and a) and a poorly grown aggregate (B and b) were fixed and sectioned. The sections were stained successively with anti-GFP mouse monoclonal antibody and alkaline phosphatase-conjugated antimouse IgG antibody (a and b), followed by staining with hematoxylin and eosin (A and B). Arrows show immunohistochemically positive cells. Scale bars represent 20 μm. ESG, early secondary spermatogonia; LSG, late-secondary spermatogonia; PC, primary spermatocytes.