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

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

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

Orchidectomy Upregulates While Testosterone Treatment Downregulates

the Expression of Ornithine Aminotransferase Gene in the Mouse Kidney 127

Fig. 7. Time course effect of testosterone on the expression of ornithine aminotransferase gene in the mouse kidney. A single dose of testosterone was injected to castrated-male mice. Mice were killed 8, 24, 28, and 32 hrs after the hormonal-treatment. Untreated castrated mice were used as controls. The values are expressed as means ± SE, (n=4 mice per group). 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. To simplify the Figure, a representative RT-PCR that corresponds to one mouse from each group was shown. B: OAT activity was measured in mouse kidneys. C: Plasma levels of corticosterone. D: Plasma levels of testosterone. The black bar represents the dark period (night). Differences between groups were statistically analyzed by Kruskal-Wallis test and followed by Mann-Whitney test. \*, P < 0.0209 in all

gene in the female rat kidney. As reported for the rats, the mouse kidney also exhibits a sexual dimorphism in the expression of OAT gene. Although the hormonal regulation of this gene has been poorly studied in both liver and kidney of male and female mice, it has been clearly shown that the endogenous production of testosterone was responsible for a decrease in the renal expression of OAT gene (Levillain et al., 2007). Interestingly, in contrast to the rats, ovariectomy altered neither the level of OAT protein nor OAT activity in the murine kidney (Levillain et al., 2007). Estrogens do not control the renal expression of OAT in the mouse kidney. Consequently, testosterone naturally controls and down-regulates the expression of

cases.

per group). To simplify the Figure, a representative RT-PCR that corresponds to one mouse from each group was shown. Abbreviations: sham-operated (Sham), and 11-day orchidectomized mice (11d), cyclophilin A (Cyclo A), eukaryotic initiation factor eIF4-E (eIF4-E), and oil + testosterone (Testo). Differences between groups were statistically analyzed by Kruskal-Wallis test, *P* = 0.1574.

## **3.5 Time course changes in the expression of OAT gene**

This experiment was performed to determine the delay requested for testosterone to downregulate the expression of OAT gene in the mouse kidney. The time-course effect of testosterone on the expression of OAT gene was analyzed at the transcriptional and posttranslational levels 8, 24, 28, and 32 hrs following hormone administration. Eight hours following testosterone treatment, the level of OAT mRNA was reduced by 8%, but did not reach statistical significance (Fig. 7A). In contrast, 24, 28, and 32 hrs after testosterone replacement, the renal levels of OAT mRNA were decreased by 45%, 40%, and 36%, respectively (Kruskal-Wallis *P* < 0.0028 followed by Mann Whitney, *P* < 0.0209 for each case). The pattern of OAT activity along this time-course study strongly resembled that of OAT mRNA except that the decrease in OAT activity was about 2-fold lower than that of OAT mRNA (Fig. 7B). Indeed, 24, 28, and 32 hrs after testosterone administration, OAT activities were decreased by 20%, 21%, and 23%, respectively (Kruskal-Wallis *P* < 0.0071 followed by Mann Whitney, *P* < 0.0209 for each case). The efficiency of the hormonal treatment was checked by measuring testosteronemia. Our results show that testosteronemia was very high 8 hrs after a single injection of the hormone and regularly decreased over the experimental period. Thirty-two hours after the treatment, testosteronemia was about 2-fold higher than the physiological concentration of testosterone reported for control male mice (Levillain et al., 2007). Corticosteronemia were quantified in the same mice to determine whether testosteronemia modified the plasma level of corticosterone. A high concentration of corticosterone was detected 8 hrs after testosterone treatment (Fig. 7C). One day later (24 and 28 hrs), the plasma level of corticosterone were decreased by 3-fold whereas at 32 hrs, it increased again by 2-fold (Fig. 7D). Unfortunately, given that it was impossible to have access to the animal room during the night, no mice could be sampled during the dark period.
