Figure 1.
Expression of Nanog in testis of various mammalian species as determined by RT-PCR.
(A) Expression of Nanog in mouse testis of the Black 6 strain (BL/6) and the SV129 strain (SV129) (B) Expression of eGFP in testis of a TNG mouse; (C) Expression of NANOG in dog and pig testes; (D) Expression of NANOG in human testis; in all RT-PCR experiments, amplicons were of the expected sizes (See also Table 2) and products were identified by sequence analysis. Abbreviations: TNG = testis cDNA from Nanog eGFP reporter mouse; Cf = Canis familiaris, Ss = Sus scrofa, Hs = Homo sapiens; + = cDNA synthesis reaction performed with reverse transcriptase, − = minus reverse transcriptase control.
Figure 2.
Expression of NANOG and eGFP in mouse testes sections as detected by immunohistochemistry.
(A–C) Expression of NANOG in mouse testis at different stages of spermatogenesis; Roman figures in seminiferous tubules represent the stage of the epithelial cycle; (D) Negative control section from which the primary antibody was omitted in the staining procedure; (E) Expression of eGFP in testis sections of a Nanog eGFP reporter; (F) eGFP staining on testis sections of a non-transgenic mouse, which served as a negative control for the staining in figures E. Abbreviations: AS = type A spermatogonia, P = Pachytene spermatocyte, T = Spermatid, ET = elongating spermatids, S = Sertoli cell, * = non-specific binding to interstitial cells. Sections displayed in panels A–D were fixed in Bouins fixative; sections in panels E and F were fixed in formalin. Formalin fixation interfered with immunostaining of the seminiferous tubules.
Figure 3.
Models for the dynamic expression of NANOG in the epithelial cycle of murine (top) and canine (bottom) spermatogenesis.
Each column represents the combination of different cell types that are present in seminiferous tubules at that specific stage. Roman figures = stage of the epithelial cycle, In = intermediate spermatogonia, B = type B spermatogonia, Pl = pre-leptotene stage, L = leptotene stage, Z = zygotene stage, P = pachytene stage, D = diplotene stage, 2nd = generation of secondary spermatocytes, 1–16 = steps in spermiogenesis. Cell types that express NANOG are outlined in red and cell types that do not express NANOG have black and grey symbols.
Figure 4.
Expression of NANOG in dog testis as determined by immunofluorescence.
(A–F) NANOG expression in differentiating male germ cells. Roman figures in panels represent the stage of the canine epithelial cycle (G–I) high magnification image of section in which the primary antibody was replaced by blocking solution, to control for non-specific binding of the secondary antibody; (J–L) image of testis section that was incubated with a rabbit IgG isotype control, to control for non-specific binding of the primary antibody.
Figure 5.
Composite image of immunoblot results for NANOG on lysates of mouse ES cells and dog and pig testes. On the right is a Benchmark protein ladder (Invitrogen) and the indicated molecular weights for each band. Abbreviations: mES = mouse embryonic stem cells, Cf = Canis familiaris, Ss = Sus scrofa.
Figure 6.
Expression of NANOG in pig testis as determined by immunofluorescence.
(A–C) NANOG expression in differentiating male germ cells; (D–F) high magnification image of NANOG expression in pachytene spermatocytes; (G–I) image of section in which the primary antibody was replaced by blocking solution, to control for non-specific binding of the secondary antibody; (J–K) representative high magnification image of testis section that was incubated with a rabbit IgG isotype control, to control for non-specific binding of the primary antibody. Dashed lines in panels A–C and G–I mark the boundaries of the tubules.
Figure 7.
Expression of NANOG in human testis as determined by immunofluorescence in paraffin-embedded sections.
(A–C) NANOG expression in differentiating male germ cells; dashed lines mark the boundaries of tubules.
Table 1.
Primers used for RT-PCR and sequence analysis.
Table 2.
Antibodies used for immuno applications.