Table 1.
List of RT-PCR and quantitative RT-PCR primers
Figure 1.
The expression of germ cell marker genes in tissues of the cynomolgus monkey.
The expression of germ cell marker genes in monkey ES cells and tissues was examined using an RT-PCR analysis. GAPDH was used as an internal control.
Figure 2.
Morphological features of cynomolgus monkey testes, and expression of VASA protein.
(A–C) Sections of testes of 3-year-old (A) and 5-year-old monkeys (B,C) were stained with hematoxylin and eosin. Sg, spermatogonium; P, pachytene spermatocyte; Sd, spermatid. (D–Q) Expression of VASA, DAZL, and SCP1 proteins during spermatogenesis in cynomolgus monkeys. Sections of 3-year-old (D,E) and 5-year old testes (F–Q) were examined by immunostaining. The nuclei were stained with Hoechst 33258. Merged images were also shown (E; VASA, red; Hoechst, white), (H, I; VASA, red; DAZL, green; Hoechst, white), (L,M; VASA, red; SCP1, green; Hoechst, white), (P,Q; DAZL, red; SCP1, green; Hoechst, white). VASA expression was observed in spermatogonia in the 3-year-old testis (E, red), and in spermatogonia (open arrowhead), spermatocytes (arrow), and early spermatids (arrowhead) in the 5-year-old testis (I, M, red). The expression of VASA, DAZL, and SCP1 proteins was all detected in spermatocytes (arrow; I, M, Q). The dotted lines indicate the basement membranes of the seminiferous tubules. The scale bar is 25 µm.
Figure 3.
The expression of germ cell marker genes in ES cells of cynomolgus monkey during EB formation.
(A) The expression patterns of germ cell marker genes in monkey testis (5 years old), mouse embryonic fibroblast (MEF), monkey ES cells (ES), and developing EBs (days 3, 7, 14, 21, and 28) were examined using an RT-PCR analysis. GAPDH was used as an internal control. (B–N) An immunocytochemical analysis of ES cells and day 14 EBs. ES cells were doubly immunostained with anti-OCT-4 and anti-VASA antibodies (B). Left column, OCT-4; middle column, VASA; right column, OCT-4 (green) and VASA (red) merged with Hoechst 33258 (white) staining. The sections of day 14 EBs were doubly immunostained with anti-SSEA1 and anti-OCT-4 (C–H) antibodies, or anti-OCT-4 and anti-VASA (I–N) antibodies. The expression of each protein is shown in left and middle columns. Merged images with Hoechst 33258 (white) staining are also shown in right column (E, H; OCT-4, red; SSEA1, green; K, N; OCT-4, green; VASA, red). The scale bars are 100 µm (B) and 25 µm (C–N), respectively.
Figure 4.
The effect of mouse testicular and ovarian cell-conditioned media on germ cell differentiation in monkey EBs.
(A) The VASA expression in ES cells (day 0) and EBs (days 7, 14, 21, and 28) in non-conditioned medium, testicular, or ovarian cell-conditioned medium was compared using quantitative RT-PCR. The expression levels of VASA in treated EBs are shown relative to VASA expression in ES cells (day 0). The data represent the means±standard deviation from triplicate PCR assays. (B) Expression of SCP1 and SCP3 in monkey testis (5 years old), ES cells (ES), and developing EBs (days 14, 21, and 28) in non-conditioned medium, testicular, or ovarian cell-conditioned medium was examined using RT-PCR. GAPDH was used as an internal control. Statistical significance was tested by Student's t-test. Asterisk, P<0.01.
Figure 5.
The effect of BMP4, RA and SCF on germ cell differentiation in monkey EBs.
(A) VASA expression in EBs cultured in the presence or absence of BMP4 (100 ng/ml), RA (1 µM), and SCF (100 ng/ml) was compared using quantitative RT-PCR. The expression levels of VASA in treated EBs are shown relative to VASA expression in ES cells (day 0). The data represent the means±standard deviation from triplicate PCR assays. (B) The expression of SCP1 and SCP3 in monkey testis (5 years old), ES cells (ES), and developing EBs (days 14, 21, and 28) in the presence or absence of BMP4 (100 ng/ml), RA (1 µM), and SCF (100 ng/ml) was examined using RT-PCR. GAPDH was used as an internal control. Statistical significance was tested by Student's t-test. Asterisk, P<0.01.