Table 1.
Oligonucleotides for specific primers.
Fig 1.
Profiles of gonadal development in control fish and chemical-induced sex change.
(A) Control fish: Approximately 100% juvenile fish gonads were differentiated to ovary and were maintained in femaleness. Fish were altered in the sexual phase from femaleness to maleness until they reached a large body size. (B) AI (aromatase inhibitor)- or MT (methyltestosterone)-treated fish: After a few months of AI or MT administration, the ovary was completely regressed, and a functional male was found (female-to-male sex change). This AI- or MT-induced maleness was transient (passive maleness), and the reversible sex change (from male-to-female) was found after AI or MT withdrawal. Eight different stages were classified during bi-directioanl sex change, including status 1 (undifferentiated gonad), status 2 (differentiated ovary), status 3 (primary oocyte stage in femaleness), status 4 (vitrellogenic oocyte stage in femaleness), status 5 (transition phase of female-to-male sex change), status 6 (initial phase of female-to-male sex change), status 7 (terminal phase of female-to-male sex change), and status 8 (transition phase of male-to-female sex reversal). S, status; Undiff, undifferentiated gonad; Diff, differentiation.
Fig 2.
Male-related gene expression profiles during female-to-male sex change.
(A-E) According to the histological events, 5 different stages were used for the gene expression pattern in femaleness (A and B), transition phase (C), and maleness (D and E). (A) Primary oocyte stage. (B) Vitellogenic oocyte stage. (C) Regressed oocyte stage. (D) Initial phase of the female-to-male sex change. (E) Terminal phase of the female-to-male sex change. (F-K) Three different categories were used to analyze the gene expression profiles, including the male germ cell marker, dmrt1 (F) and Sertoli cell marker, sox9 (G); key enzyme for 11-KT synthesis, cyp11b2 (H); amh (I); amhr2 (J); and enzyme for E2 synthesis, cyp19a1a (K). IP, initial phase; PO, primary oocyte; RO, regressed oocyte; SG, spermatogonia; SP, sperm; TP, terminal phase; VO, vitellogenic oocyte.
Fig 3.
Location of Amh in the testes.
(A) Western blotting of grouper testicular and ovarian protein extracts using anti-Amh antiserum and anti-Actin antiserum. The black arrowhead denotes Amh protein. (B and C) Immunohistochemical staining of testes using anti-Amh antiserum. Black arrowheads indicate the positive signal (brown color) of anti-Amh antiserum. (D and E) Immunofluorescent staining of testes using anti-Amh antiserum and anti-Vasa antiserum. Vasa is a germ cell marker. White arrowheads indicate a positive signal (green color) of anti-Amh antiserum. OT, ovarian tissue; RO, regressed oocyte, SC, spermatocyte; SG, spermatogonia, SP, sperm; TT, testicular tissue.
Fig 4.
Immunohistochemical analysis of Amh in the gonad during the female-to-male sex change.
Immunohistochemical staining of gonads at different stages using anti-Amh antiserum. (A) Vitellogenic oocyte stage. (B) Regressed oocyte stage. (C) Initial phase of the female-to-male sex change. (D) Terminal phase of the female-to-male sex change. (E) Western blotting of grouper gonad protein extracts using anti-Amh antiserum. Black arrowheads indicate the positive signal (brown color) of anti-Amh antiserum. EPO, early primary oocyte; Fc, follicle cell; IP, initial phase; PO, primary oocyte; RO, regressed oocyte; VO, vitellogenic oocyte; SC, spermatocyte; SG, spermatogonia; SP, sperm; TP, terminal phase.
Fig 5.
amh and amhr2 expression profiles during bi-directional sex change.
Immunohistochemical staining of gonads using anti-Pcna antiserum (A, B, and D) and anti-Brdu antiserum (C and E). Pcna as a proliferating marker and Brdu-incorporated cells revealed high proliferating activity. (A) Ovary at 0 day before AI or MT administration. (B) Testes at 12 weeks after AI administration. (C) Regressed testes at 3 weeks after AI withdrawal. (D) Testes at 12 weeks after MT administration. (E) Regressed testes at 3 weeks after MT withdrawal. amh and amhr2 expression profiles in different statuses in AI-treated fish (F and G) and MT-treated fish (H and I). Asterisk indicates a Student’s t-test (P < 0.05). Student’s t-test was also conducted to compare the significant differences (P < 0.05) between the treatments. L, central lumen; OG, oogonia; PO, primary oocyte; SG, spermatogonia; SGL, type A spermatogonia-like cell; SP, sperm.
Fig 6.
Male-related gene expression profiles during reversible male-to-female sex change.
MT-induced maleness is a transient status. Male characteristics were diminished after methyltestosterone (MT) withdrawal (1–2 weeks), and then a male-to-female sex change occurred. Expression of male-related genes dmrt1 (A), sox9 (B), cyp11b2 (C), amh (D), and amhr2 (E) during the initial phase of male-to-female sex change in MT-terminated fish (day 0 to day 14). Superscript letters indicate one-way ANOVA and Student-Newman-Keuls multiple test (P < 0.05).
Fig 7.
Immunohistochemical analysis of Amh in the gonad during reversible male-to-female sex change.
Immunohistochemical staining of gonads at different stages using anti-Amh antiserum. (A and B) Primary oocyte stage in the female gonad. (C and D) Initial phase of the male-to-female reversible sex change in the gonad after a week of methyltestosterone (MT) administration. (E-F) Amh signals in the early stage (4 of 6 fish) of reversible male-to-female sex change after two weeks of MT withdrawal in MT-induced males (3 months treatment). (G-H) No Amh signals in the later stage (2 of 6 fish) of reversible male-to-female sex change after two weeks of MT withdrawal in MT-induced males (3 months treatment). The black arrowheads indicate the positive signal (brown color) of anti-Amh antiserum. CT, connective tissue; L, central lumen; PO, primary oocytes; OG, oogonia; SG, spermatogonia; SGL, type A spermatogonia-like cells.