Table 1.
Media used with growth factors in total testicular cell culture.
Table 2.
Primers used for RT-PCR.
Figure 1.
Histological analysis and spermatogonia detection in 12-month-old beagle testes.
Panels A, B, C, and D are hematoxylin-eosin–stained testis sections. Spermatogenetic cells are detected in 12-month-old beagle testes. Testis sections were stained with PGP9.5 (E) and DAZL (F) antibodies. Black arrows indicate PGP9.5-positive spermatogonia in panel E and white arrows indicate DAZL-positive spermatocytes in panel F. Scale bars are 20 µm in all panels. EST, elongated spermatid; RST, round spermatid; SC, Sertoli cell; SCT, spermatocyte; SG, spermatogonia; SZ, spermatozoa. Magnification is ×1000 (panels A, B, C, and D) and ×400 (panels E and F).
Figure 2.
Histological analysis and spermatogonia detection in 3- and 2-month-old beagle testes.
Panels A and C are hematoxylin-eosin–stained testis sections in 3- and 2-month-old beagle testes, respectively. Only spermatogonia exist in 3- and 2-month-old beagle testes. PGP9.5-positive spermatogonia are shown in panel B (3-month-old beagle testes) and D (2-month-old beagle testes). Arrows indicate PGP9.5-positive spermatogonia. Scale bars indicate 20 µm in all panels. IC, interstitial cells; SC, Sertoli cell; SG, spermatogonia. Magnification is ×400 in all panels.
Figure 3.
Body weight, size of the seminiferous tubules, and number of PGP9.5-positive spermatogonia in 2-, 3-, and 12-month-old beagle testes.
(A) Body weight, (B) seminiferous tubule diameter, and (C) number of PGP9.5-positive spermatogonia. Diameters of rounded seminiferous tubules were calculated by Stereo Investigator Version 7.5, and the numbers of PGP9.5-positive spermatogonia were counted from the images of 2-, 3-, and 12-month-old beagle testis sections taken at ×400 magnification (C). (* P<0.05).
Figure 4.
Colony formation in stempro-34 and DMEM-FBS media with growth factors.
Total testicular cells were cultured for 7 days in (A) stempro-34, (B) DMEM- 1%FBS, (C) DMEM- 5%FBS, and (D) DMEM- 1%FBS. Combinations of growth factors were added in each media. Arrowheads indicate the colonies formed in specific media conditions. Scale bars indicate 100 µm in all panels. GDNF, glial cell-derived neurotrophic factor; bFGF, basic fibroblast growth factor; LIF, leukemia inhibitory factor; EGF, epidermal growth factor. Magnification is ×100 in all panels.
Figure 5.
Characterization of colonies cultured in stempro-34 and DMEM-5%FBS media conditions at day 7.
Colonies cultured in stempro-34 supplemented with GDNF, FGF, LIF, and EGF (A, B, C, D, and E) and DMEM-5% FBS supplemented with GDNF, FGF, LIF, and EGF (F, G, H, I, and J) were analyzed. AP-staining results are in A and F. Colonies stained with PGP9.5 antibody are in B and G. Nuclear staining is shown in panels C and H. Bright-field images are shown in panels D and I. Collected colonies for PCR analysis are shown in panels E and J. Arrowheads indicate the colonies. RT-PCR and real-time PCR were performed on colonies and 2-month-old beagle testes (panels K and L), respectively, with OCT4, NANOG, PLZF, PGP9.5, GFRα-1, LHR, and GATA4 primers, as shown in Table 2. Magnification is ×200 in panels A–D and F–I, and ×100 in panels E and J. MOBT, month-old beagle testes. (* P<0.05).