**3.2 Influence of orchidectomy on the expression of OAT gene**

A complete analysis of the expression of OAT gene in the mouse kidney was undertaken by analyzing the levels of mRNA and protein, and measuring OAT activity. The level of OAT

Orchidectomy Upregulates While Testosterone Treatment Downregulates

the Expression of Ornithine Aminotransferase Gene in the Mouse Kidney 123

The values are expressed as means ± SE. A: The level of OAT mRNA were analyzed by semiquantitative RT-PCR. The amplified cDNAs were separated by agarose-gel electrophoresis. Band intensities were quantified and reported relative to the cyclophilin A band (n=3 mice per group). B: Immunoblot assessing the abundance of OAT and β-actin proteins in mouse kidneys. Immunoblots were loaded with samples of 100 µg soluble proteins (n=6 mice per group). β-actin was used as control of protein loading and transfer. C: OAT activity was measured in mouse kidneys (n= 4, 3, 3, 3 mice per group, respectively). To simplify the Figure, a representative RT-PCR or immunoblot that corresponds to one mouse from each group was shown. Abbreviations: control (Cont), sham-operated (Sham), and 11- and 18-day orchidectomized mice (11d and 18d, respectively), cyclophilin A (Cyclo A), ornithine aminotransferase (OAT), and absorbance (Abs). Differences between groups were statistically

analyzed by Kruskal-Wallis test and followed by Mann-Whitney test. \*, *P* < 0.05 or less.

Fig. 4. Relative levels of ornithine aminotransferase mRNA in mouse kidneys.

Number of mice used: 11, 6, 9, 7, 6, and 6, respectively.

**3.3 Influence of testosterone on the expression of OAT gene** 

The values are expressed as means ± SE. The relative mRNA levels of OAT were determined by qPCR in the kidneys of male (M), sham-operated (Sham), 11- and 18-day orchidectomized mice (11d and 18d, respectively), castrated mice treated with oil as solvant (Oil), and castrated mice supplemented with testosterone dissolved in oil (Testo). Abbreviations: ornithine aminotransferase (OAT), and hypoxanthine guanine phosphoribosyl transferase (HPRT). Differences between groups were statistically analyzed by Kruskal-Wallis test (*P* < 0.0001) and followed by Mann-Whitney test. \*, *P* < 0.0039 or less.

Given that orchidectomy provoked a sharp increase in the expression of OAT gene in the mouse kidney, we hypothesized that androgens control and down-regulate the expression of this gene. To verify our hypothesis, castrated male mice were subjected to testosteronetreatment. An additional group of castrated mice was subjected to sesame oil-treatment inasmuch as oil was used as a solvant to dissolve testosterone. The expression of OAT gene was analyzed at the transcriptional and traductional levels and OAT activity was measured. In

mRNA, protein, and enzyme activity did not differ between control and sham-operated male mice. Surgery did not affect the expression of OAT gene in the mouse kidney (Fig. 3). In contrast, orchidectomy provoked a sharp increase in OAT mRNA, protein, and enzyme activity. In details, in castrated mice killed 11 day after the surgery, the level of OAT mRNA, OAT protein, and OAT activity were increased by 3.95-fold (Kruskal-Wallis, *P* < 0.035 followed by Mann Whitney, *P* < 0.0495), 2.13-fold (Kruskal-Wallis, *P* < 0.0004 followed by Mann Whitney, *P* < 0.0039), and 2.27–fold (Kruskal-Wallis, *P* < 0.018 followed by Mann Whitney, *P* < 0.05), respectively as compared to the sham-operated mice (Fig. 3). When orchidectomized mice were killed 18 days after the operation, OAT gene remained still highly expressed, nevertheless, its expression had a tendancy to be higher than at day 11. The level of OAT mRNA was 4.44-fold higher than that of the sham-operated mice (Mann Whitney, *P* < 0.0495), but did not differ from that of the mice killed on day 11. Similarly, the mean value of OAT protein was higher on day 18 than on day 11, but it did not reach the statistical level of significance (*P* < 0.0547). In contrast, OAT activity was significantly higher on day 18 than day 11 as compared to the sham-operated mice (Mann Whitney, *P* < 0.0495). The level of OAT mRNA was quantitated either by RT-PCR (Fig. 3) or by q-PCR (Fig. 4). The two methods gave similar results and patterns (Figs. 3 and 4). Different house-keeping genes were used to prove that the effect of castration were independent of the gene of reference. Orchidectomy provoked a 4.9- and 4.7-fold increase in the level of OAT mRNA in 11d- and 18d-castrated mice as compared to the sham-operated male mice. (Fig. 4; Kruskal-Wallis, *P* < 0.0001 followed by Mann Whitney, *P* < 0.0027 or less in both cases).

Fig. 3. Effect of orchidectomy on the expression of ornithine aminotransferase gene in the mouse kidney.

mRNA, protein, and enzyme activity did not differ between control and sham-operated male mice. Surgery did not affect the expression of OAT gene in the mouse kidney (Fig. 3). In contrast, orchidectomy provoked a sharp increase in OAT mRNA, protein, and enzyme activity. In details, in castrated mice killed 11 day after the surgery, the level of OAT mRNA, OAT protein, and OAT activity were increased by 3.95-fold (Kruskal-Wallis, *P* < 0.035 followed by Mann Whitney, *P* < 0.0495), 2.13-fold (Kruskal-Wallis, *P* < 0.0004 followed by Mann Whitney, *P* < 0.0039), and 2.27–fold (Kruskal-Wallis, *P* < 0.018 followed by Mann Whitney, *P* < 0.05), respectively as compared to the sham-operated mice (Fig. 3). When orchidectomized mice were killed 18 days after the operation, OAT gene remained still highly expressed, nevertheless, its expression had a tendancy to be higher than at day 11. The level of OAT mRNA was 4.44-fold higher than that of the sham-operated mice (Mann Whitney, *P* < 0.0495), but did not differ from that of the mice killed on day 11. Similarly, the mean value of OAT protein was higher on day 18 than on day 11, but it did not reach the statistical level of significance (*P* < 0.0547). In contrast, OAT activity was significantly higher on day 18 than day 11 as compared to the sham-operated mice (Mann Whitney, *P* < 0.0495). The level of OAT mRNA was quantitated either by RT-PCR (Fig. 3) or by q-PCR (Fig. 4). The two methods gave similar results and patterns (Figs. 3 and 4). Different house-keeping genes were used to prove that the effect of castration were independent of the gene of reference. Orchidectomy provoked a 4.9- and 4.7-fold increase in the level of OAT mRNA in 11d- and 18d-castrated mice as compared to the sham-operated male mice. (Fig. 4; Kruskal-

Wallis, *P* < 0.0001 followed by Mann Whitney, *P* < 0.0027 or less in both cases).

Fig. 3. Effect of orchidectomy on the expression of ornithine aminotransferase gene in the

mouse kidney.

The values are expressed as means ± SE. A: The level of OAT mRNA were analyzed by semiquantitative RT-PCR. The amplified cDNAs were separated by agarose-gel electrophoresis. Band intensities were quantified and reported relative to the cyclophilin A band (n=3 mice per group). B: Immunoblot assessing the abundance of OAT and β-actin proteins in mouse kidneys. Immunoblots were loaded with samples of 100 µg soluble proteins (n=6 mice per group). β-actin was used as control of protein loading and transfer. C: OAT activity was measured in mouse kidneys (n= 4, 3, 3, 3 mice per group, respectively). To simplify the Figure, a representative RT-PCR or immunoblot that corresponds to one mouse from each group was shown. Abbreviations: control (Cont), sham-operated (Sham), and 11- and 18-day orchidectomized mice (11d and 18d, respectively), cyclophilin A (Cyclo A), ornithine aminotransferase (OAT), and absorbance (Abs). Differences between groups were statistically analyzed by Kruskal-Wallis test and followed by Mann-Whitney test. \*, *P* < 0.05 or less.

Fig. 4. Relative levels of ornithine aminotransferase mRNA in mouse kidneys.

The values are expressed as means ± SE. The relative mRNA levels of OAT were determined by qPCR in the kidneys of male (M), sham-operated (Sham), 11- and 18-day orchidectomized mice (11d and 18d, respectively), castrated mice treated with oil as solvant (Oil), and castrated mice supplemented with testosterone dissolved in oil (Testo). Abbreviations: ornithine aminotransferase (OAT), and hypoxanthine guanine phosphoribosyl transferase (HPRT). Differences between groups were statistically analyzed by Kruskal-Wallis test (*P* < 0.0001) and followed by Mann-Whitney test. \*, *P* < 0.0039 or less. Number of mice used: 11, 6, 9, 7, 6, and 6, respectively.

#### **3.3 Influence of testosterone on the expression of OAT gene**

Given that orchidectomy provoked a sharp increase in the expression of OAT gene in the mouse kidney, we hypothesized that androgens control and down-regulate the expression of this gene. To verify our hypothesis, castrated male mice were subjected to testosteronetreatment. An additional group of castrated mice was subjected to sesame oil-treatment inasmuch as oil was used as a solvant to dissolve testosterone. The expression of OAT gene was analyzed at the transcriptional and traductional levels and OAT activity was measured. In

Orchidectomy Upregulates While Testosterone Treatment Downregulates

**3.4 Influence of testosterone on the expression of eIF4-E gene** 

Whitney test. \*, *P* < 0.05 or less.

significance (Fig. 6, Kruskal-Wallis, *P* < 0.1574).

the Expression of Ornithine Aminotransferase Gene in the Mouse Kidney 125

The values are expressed as means ± SE. A: The level of OAT mRNA were analyzed by semi-quantitative RT-PCR. The amplified cDNAs were separated by agarose-gel electrophoresis. Band intensities were quantified and reported relative to the cyclophilin A band (n=3 mice per group). B: Immunoblot assessing the abundance of OAT and G3PDH proteins in mouse kidneys. Immunoblots were loaded with samples of 100 µg soluble proteins (n=3 mice per group). G3PDH was used as as control of protein loading and transfer. C: OAT activity was measured in mouse kidneys (n=4 mice per group). D: Plasma levels of corticosterone (n = 9, 10, 10, and 9 mice, respectively). To simplify the Figure, a representative RT-PCR or immunoblot that corresponds to one mouse from each group was shown. Abbreviations: sham-operated (Sham), and 11-day orchidectomized mice (11d), cyclophilin A (Cyclo A), ornithine aminotransferase (OAT), G3PDH (glyceraldehyde-3 phosphate dehydrogenase), Testo (oil + testosterone), and absorbance (Abs). Differences between groups were statistically analyzed by Kruskal-Wallis test and followed by Mann-

The eukaryotic initiation factor eIF4-E has been reported to be rate-limiting for OAT translation (Fagan et al., 1991). We analyzed whether the level of eIF4-E mRNA was altered by orchidectomy and testosterone replacement. Our data show that, in the mouse kidney, the level of eIF4-E mRNA had a tendancy to be decreased by orchidectomy and enhanced by testosterone replacement. Nevertheless, the difference did not reach statistical

Fig. 6. Effect of testosterone treatment on the expression of eIF4-E gene in the mouse kidney. The values are expressed as means ± SE. The levels of eIF4-E mRNA were analyzed by semiquantitative RT-PCR. The amplified cDNAs were separated by agarose-gel electrophoresis. Band intensities were quantified and reported relative to the cyclophilin A band (n=3 mice

this new study, our results confirmed that castration of male mice provoked an overexpression of OAT gene (Fig. 5). The levels of OAT mRNA, protein, and enzyme activity were increased and strongly resemble those presented in Figure 3. However, the level of OAT mRNA was increased only by two-fold in kidneys of castrated mice of the second study, (Fig. 5) as compared to four-fold for the first study (Fig. 3). In kidneys of castrated mice treated with a single injection of sesame oil, the level of OAT mRNA, protein, and OAT activity did not differ from those of the castrated mice (Fig. 5). In contrast, a single injection of a physiological dose of testosterone decreased by 2-fold the level of OAT mRNA (Kruskal-Wallis, *P* < 0.036 followed by Mann Whitney, *P* < 0.0495), OAT protein by 1.58-fold (Kruskal-Wallis, *P* < 0.032 followed by Mann Whitney, *P* < 0.05), and OAT activity by 1.62-fold (Kruskal-Wallis, *P* < 0.0045 followed by Mann Whitney, *P* < 0.0209) (Fig. 5). The level of OAT mRNA was also quantitated either by RT-PCR (Fig. 5) or by q-PCR (Fig. 4). Different house-keeping genes were used to prove that the effect of castration were independent of the gene of reference. Again, the same pattern was found with the two methods (Figs. 4 and 5). Oil-treatment had no effect whereas testosterone-treatment provoked a 5.3-fold decrease in the level of OAT mRNA as compared to oil-treated mice. (Fig. 5; Kruskal-Wallis, *P* < 0.0001 followed by Mann Whitney, *P* < 0.0039). The plasma levels of corticosterone had a tendancy to increase after orchidectomy and decrease in testosterone-treated mice. Unfortunately, the difference did not reach statistical significance (Kruskal-Wallis, *P* < 0.2435). The plasma level of corticosterone were inversely correlated with those of testosterone (Fig. 5 and Levillain et al. 2005).

Fig. 5. Effect of testosterone treatment on the expression of ornithine aminotransferase gene in the mouse kidney.

this new study, our results confirmed that castration of male mice provoked an overexpression of OAT gene (Fig. 5). The levels of OAT mRNA, protein, and enzyme activity were increased and strongly resemble those presented in Figure 3. However, the level of OAT mRNA was increased only by two-fold in kidneys of castrated mice of the second study, (Fig. 5) as compared to four-fold for the first study (Fig. 3). In kidneys of castrated mice treated with a single injection of sesame oil, the level of OAT mRNA, protein, and OAT activity did not differ from those of the castrated mice (Fig. 5). In contrast, a single injection of a physiological dose of testosterone decreased by 2-fold the level of OAT mRNA (Kruskal-Wallis, *P* < 0.036 followed by Mann Whitney, *P* < 0.0495), OAT protein by 1.58-fold (Kruskal-Wallis, *P* < 0.032 followed by Mann Whitney, *P* < 0.05), and OAT activity by 1.62-fold (Kruskal-Wallis, *P* < 0.0045 followed by Mann Whitney, *P* < 0.0209) (Fig. 5). The level of OAT mRNA was also quantitated either by RT-PCR (Fig. 5) or by q-PCR (Fig. 4). Different house-keeping genes were used to prove that the effect of castration were independent of the gene of reference. Again, the same pattern was found with the two methods (Figs. 4 and 5). Oil-treatment had no effect whereas testosterone-treatment provoked a 5.3-fold decrease in the level of OAT mRNA as compared to oil-treated mice. (Fig. 5; Kruskal-Wallis, *P* < 0.0001 followed by Mann Whitney, *P* < 0.0039). The plasma levels of corticosterone had a tendancy to increase after orchidectomy and decrease in testosterone-treated mice. Unfortunately, the difference did not reach statistical significance (Kruskal-Wallis, *P* < 0.2435). The plasma level of corticosterone were inversely

Fig. 5. Effect of testosterone treatment on the expression of ornithine aminotransferase gene

in the mouse kidney.

correlated with those of testosterone (Fig. 5 and Levillain et al. 2005).

The values are expressed as means ± SE. A: The level of OAT mRNA were analyzed by semi-quantitative RT-PCR. The amplified cDNAs were separated by agarose-gel electrophoresis. Band intensities were quantified and reported relative to the cyclophilin A band (n=3 mice per group). B: Immunoblot assessing the abundance of OAT and G3PDH proteins in mouse kidneys. Immunoblots were loaded with samples of 100 µg soluble proteins (n=3 mice per group). G3PDH was used as as control of protein loading and transfer. C: OAT activity was measured in mouse kidneys (n=4 mice per group). D: Plasma levels of corticosterone (n = 9, 10, 10, and 9 mice, respectively). To simplify the Figure, a representative RT-PCR or immunoblot that corresponds to one mouse from each group was shown. Abbreviations: sham-operated (Sham), and 11-day orchidectomized mice (11d), cyclophilin A (Cyclo A), ornithine aminotransferase (OAT), G3PDH (glyceraldehyde-3 phosphate dehydrogenase), Testo (oil + testosterone), and absorbance (Abs). Differences between groups were statistically analyzed by Kruskal-Wallis test and followed by Mann-Whitney test. \*, *P* < 0.05 or less.
