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Gen Comp Endocrinol
2007 Jan 01;1531-3:280-8. doi: 10.1016/j.ygcen.2007.01.041.
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Aromatase, steroid-5-alpha-reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny.
Urbatzka R
,
Lutz I
,
Kloas W
.
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The key enzymes involved in the production of endogenous sex steroids are steroid-5-alpha-reductase and aromatase converting testosterone (T) into dihydrotestosterone (DHT) and into estradiol (E2), respectively. To gain more insights into the molecular mechanisms of sexual differentiation of amphibians, we determined the mRNA expression of steroid-5-alpha-reductase type1 (Srd5a1), type2 (Srd5a2) and aromatase (Aro) during ontogeny starting from the egg and ending after completion of metamorphosis in Xenopus laevis. Expression of all three enzymes was measured by means of semi-quantitative RT-PCR, determining for the first time Srd5a1 and Srd5a2 mRNA expression in amphibians. mRNA was analyzed in whole body homogenates from stage 12 to 48, while brain and gonads with kidney were studied separately from stage 48 to 66. Different ontogenetic mRNA expression patterns were observed for all genes analyzed, revealing early mRNA expression of Srd5a1 already in the egg at stage 12 whereas Srd5a2 and Aro was detected at stage 39. Sex-specific mRNA expressions of Srd5a2 and of Aro were determined in the gonads with kidney but not in brain. Srd5a2 was two-fold higher expressed in testes than in ovaries while Aro mRNA was ten-fold higher in ovaries. No gender-specific mRNA expression was observed for Srd5a1 in gonads and in brain. The ontogenetic patterns of Aro, Srd5a1 and Srd5a2 suggest that these genes are involved in sexual differentiation of gonads and brain already in early developmental stages. Especially in gonads Srd5a2 seems to be important for physiological regulation of testis development while Aro is associated with the development of ovaries.
Fig. 1.
Qualitative expression of aromatase (Aro), steroid-5-alpha-reductase type 1 (Srd5a1), type 2 (Srd5a2) and elongation factor-1α (EF) mRNA in different tissues of male and female tadpoles of X. laevis are shown on an agarose gel. Expression of mRNA was analyzed at developmental stage 59 according to Nieuwkoop and Faber (1994) in gonads (G), heart (H), spleen (S), brain (B), liver (L) and kidney (K). Samples were over-amplified and cycle numbers for PCR reactions were 36 for Aro, Srd5a1 and Srd5a2, and 20 for EF. Determinations of Aro, Srd5a1, Srd5a2 and EF demonstrate specific product lengths of 274, 308, 146 and 285 bp, respectively. The molecular weight marker indicates distances of 100 bp DNA.
Fig. 2.
Relative mRNA expression of house-keeping genes, elongation factor-1α (EF), β-2-microglobulin (B2M), ornithine decarboxylase (ODC) and histone 4 (H4), in X. laevis tadpoles are shown during the first analyzed developmental stages according to Nieuwkoop and Faber (1994) from stage 12 to 48 of whole body homogenates (WB). Additionally, at stage 48 tadpoles were dissected in brain (B), gonads (G) and remaining tissue (R) and the mRNA expression was analyzed. Differences in EF and H4 mRNA expression were significant (p < 0.05) in the developmental period between stages 12 and 48, and therefore B2M was chosen as house-keeping gene for this period being more constantly expressed than ODC.
Fig. 3.
Relative mRNA expression of aromatase (Aro), steroid-5α-reductase type 1 (Srd5a1) and type 2 (Srd5a2) in whole body homogenates of X. laevis tadpoles during early stages of development (stage 12–48). Additionally, at stage 48 tadpoles were dissected in brain (B), gonads with kidney (G) and remaining tissue (R) and mRNA expression of Aro, Srd5a1 and Srd5a2 was analysed. Values are expressed as means ± SD derived from six individuals at each developmental stage. Densitometric values were normalized by the housekeeping gene β-2-microglobulin (B2M) for the whole body homogenates and by elongation factor-1α (EF-1α) for the individual tissues at stage 48 (cf. Fig. 2). Significant differences were tested using one-Way ANOVA, Dunnett-T3, and were indicated by different letters (p < 0.05).
Fig. 4.
Relative mRNA expression of aromatase (Aro) mRNA in gonads with kidney (G, upper panel) and in brain (B, lower panel) of tadpoles of X. laevis during ontogeny. Values are expressed as means ± SD derived from six individuals at each developmental stage. Densitometric values were normalized by the housekeeping gene elongation factor-1α (EF-1α) for all samples. Samples were subdivided into sample sets and interconnected for the presentation of the data in a comprehensive figure. Aro mRNA expression is presented in relation to stage 48 of the gonads and of the brain, respectively, that was arbitrary set to the reference value 1. Statistical significant differences between the mRNA levels of the various stages were tested within the sample sets and between male and female tadpoles using one-way ANOVA followed by Dunnett-T3 or Tukey test, depending on the homogeneity of variance of the data. Significant differences between the developmental stages were indicated by different letters (p < 0.05) and between males and females by asterisks (p < 0.05).
Fig. 5.
Relative mRNA expression of steroid-5-alpha-reductase type 1 (Srd5a1) mRNA in gonads with kidney (G, upper panel) and in brain (B, lower panel) of tadpoles of X. laevis during ontogeny corresponding to Fig. 4. Values are expressed as means ± SD derived from six individuals at each developmental stage. Significant differences between the developmental stages were indicated by different letters (p < 0.05).
Fig. 6.
Relative mRNA expression of steroid-5α-reductase type 2 (Srd5a2) mRNA in gonads with kidney (G, upper panel) and in brain (B, lower panel) of tadpoles of X. laevis during ontogeny corresponding to Fig. 4. Values are expressed as means ± SD derived from six individuals at each developmental stage. Significant differences between the developmental stages were indicated by different letters (p < 0.05) and between males and females by asterisks (p < 0.05).
