October 1, 2008;
Sex-specific expression of SOX9 during gonadogenesis in the amphibian Xenopus tropicalis.
To investigate the role of SOX9
gene in amphibian gonadogenesis, we analyzed its expression during male and female gonadogenesis in Xenopus tropicalis. The results showed that in both sexes SOX9
mRNA and protein were first detectable after metamorphosis when the gonads were well differentiated and remained present until the adult stage. In the testis
expression was restricted to the nucleus
of Sertoli-like cells, similarly to what has been observed in other vertebrates suggesting a conserved role in vertebrate testicular differentiation. In the ovary
, in sharp contrast with what has been observed in all vertebrates examined so far, the SOX9
protein was localized in the cytoplasm
of previtellogenic oocytes before being translocated into the nucleus
of vitellogenic oocytes suggesting an unexpected role during oogenesis. These results suggest that the SOX9
gene may not be a sex-determining gene in X. tropicalis and may play different functions in testicular and ovarian differentiation.
[+] show captions
Figure 4. Expression of SOX9 mRNA and protein in the developing male gonad of Xenopus tropicalis after metamorphosis. A-I: SOX9 expression in the male gonad after metamorphosis at (Test 4). A-C: In situ hybridization with SOX9 sense or antisense RNA probes. A: Section from male gonad at this stage hybridized with SOX9 sense probe as a control. B: Antisense probe. C: Fibronectin labeling was superimposed on the same section used for SOX9 mRNA detection to identify the hybridizing positive cells. Fibronectin outlined the testicular cords where SOX9 is expressed, whereas there is no SOX9 expression within the interstitial tissue positive for fibronectin. D: Histological cross section showing testicular cords (TC).G: High magnification of the square in D showing the supporting cells (indicated by arrowheads) which surround germ cells. E,H: Immunodetection of SOX9 protein. E: Frozen section stained with anti SOX9 antibody (green) and counterstained with Hoechst (blue). H: High magnification of the square in E shows by comparison with Fig. G that SOX9 immunostaining (green) is detected only within the nuclei of supporting cells (blue) that surround germ cells (indicated by asterisks). F: Frozen section double labeled with SOX9 (green) and laminin (read) and counterstained with Hoechst (blue), note the detection of laminin around testicular cords. I: High magnification of the square in F. Scale bars = 30 mu m in A-F; 20 mu m in G-I.
Figure 5. Expression of SOX9 mRNA and protein in the Xenopus tropicalis adult male gonad. A-D: Histological cross-section of X. tropicalis male gonad at the adult stage. A: Cross-section showing that the testis is composed of seminiferous tubules. B-D: Details of different areas within the seminiferous tubules in A showing different stages of cyst differentiation each cyst being surrounded by supporting cells (arrowheads). B: Cyst of primary spermatocytes. C: Cyst of secondary spermatocytes. D: Spermatid. E-G: In situ hybridization with the antisense mRNASox9 probe. E: Section showing that SOX9 expression is maintained in the adult male gonad. F: Detail of the region indicated by the square in E shows that SOX9 expression was detected inside seminiferous tubules only. G: The 3 beta -hydroxysteroid dehydrogenase (3- beta -HSDH) enzymatic reaction on adjacent sections showing that steroidogenic activity is located in the interstitial tissue, which surrounds the seminiferous tubules. H-M: Immunodetection of SOX9 protein in the male gonad at the same stage. H,J: Cryosection stained with anti-SOX9 antibody (green) and counterstained with Hoechst (blue). I,K: High magnifications of the portions from H, J respectively showing the SOX9 signal is restricted to nuclei of the supporting cells (indicated by arrowheads) irrespective of the stage of the germ cells. L: Overlap of images H and J. M: Overlap of images I and K. IT, interstitial tissue; SI, primary spermatocytes; SII, secondary spermatocytes; Sd, spermatid; ST, seminiferous tubules. Scale bars = 50 mu m in A,F-H,J,L, 30 mu m in B-D, 150 mu m in L, 30 mu m in I,K,M.