**Orchidectomy Upregulates While Testosterone Treatment Downregulates the Expression of Ornithine Aminotransferase Gene in the Mouse Kidney**

Olivier Levillain, Cyril Dégletagne, Dominique Letexier and Henri Déchaud1 *University Claude Bernard Lyon 1, UMR 5123 CNRS 1University Claude Bernard Lyon 1, U1060 INSERM France* 

#### **1. Introduction**

114 Basic and Clinical Endocrinology Up-to-Date

Wynne F.L. & Khalil R.A. (2003). Testosterone and coronary vascular tone: implications in

Yamada T., Fujino T., Yuhki K. *et al*. (2003). Thromboxane A2 regulates vascular tone via its

Yorek M.A., Coppey L.J., Gellett J.S. *et al*. (2002). Effect of treatment of diabetic rats with

inhibitory effect on the expression of inducible nitric oxide synthase. *Circulation*

dehydroepiandrosterone on vascular and neural function. *Am J Physiol* 283: 1067-

coronary artery disease. *J Endocrinol Invest* 26: 181-186.

108: 2381-2386.

1075.

Ornithine aminotransferase (L-ornithine: 2-oxoacid aminotransferase, OAT, EC 2.6.1.13) plays crucial physiological roles in amino acid metabolism because this enzyme is at the crossroad of several pathways including those of L-arginine, L-ornithine, L-glutamate, Lglutamine, and L-proline. Specifically, OAT catalyzes the transamination of L-ornithine in the presence of α-ketoglutarate to produce one molecule of L-glutamate and the unstable compound glutamate-γ-semialdehyde that is spontaneously converted into Δ1-pyrroline-5 carboxylate. This latter molecule is further metabolized by the enzyme pyrroline-5 carboxylate dehydrogenase into a second molecule of L-glutamate (Wakabayashi, 2004). The enzyme is expressed in many mammalian tissues including the liver, the kidney, and the intestine which exhibit the highest OAT activities (Peraino & Pitot, 1963; Herzfeld & Knox, 1968; Sanada et al., 1970; Kasahara et al., 1986; Alonso & Rubio, 1989; Levillain et al., 2007; Ventura et al., 2009). These enzymes may not only display diverse tissue-specific physiological roles, but demonstrate marked sexual differences in expression and activity.

In rat kidneys, estrogen dramatically increased the expression of OAT and is responsible for the higher levels of OAT in female than in male rat kidney (Herzfeld & Knox, 1968; Lyons & Pitot, 1977; Mueckler & Pitot, 1983; Mueckler et al., 1984; Levillain et al., 2004). The presence of thyroid hormone is required for estrogen induction. These hormones exert a synergistic effect on the expression of OAT gene (Mueckler & Pitot, 1983). The expression of OAT gene during the rat postnatal development strongly supports the sexual dimorphism of OAT in kidney, but not in liver (Herzfeld & Knox, 1968; Herzfeld & Greengard, 1969). Taken together, the expression of OAT gene in the female rat kidney is naturally upregulated in the presence of estrogen.

The expression of OAT gene in the mouse kidney has been reported by different authors who independently measured OAT mRNA and protein levels or OAT activity (Alonso & Rubio, 1989; Natesan & Reddy, 2001; Yu et al., 2003; Levillain et al., 2005; Manteuffel-Cymborowska et al., 2005; Levillain et al., 2007; Ventura et al., 2009). Strong evidences

Orchidectomy Upregulates While Testosterone Treatment Downregulates

(authorization no. 299 revised and no. BH 2009-15).

liquid nitrogen until testosterone and corticosterone measurement.

**2.3 RNA extraction and semiquantitative RT-PCR** 

**2.2 Sampling of kidneys and blood** 


further in the text.

**2.3.1 RNA extraction**

the Expression of Ornithine Aminotransferase Gene in the Mouse Kidney 117

Fig. 1. Schema of the time-course study of testosterone effect on the expression of OAT gene. Animal care complied with French regulations for the protection of animals used for experimental and other scientific purposes and with European Community regulations (Council of Europe N° 123, Strasbourg, 1985). The author (O. Levillain) is authorized by the "Direction Départementale des Services Vétérinaires" (authorization no. 69-33 and 69266391) and the local Animal Care Committee to use animals for these experiments

The renal pedicle of each kidney was clamped and the kidney was rapidly removed, decapsulated; the blood contained in each kidney was removed with blotting paper (freeblood). The kidney was placed in a sterilized Eppendorf tube, frozen, and conserved at

Blood was collected in the vena cava of all mice with a 25-gauge needle (Neolus, VWR, Limonest, France) mounted on a 1-mL syringe (Terumo, VWR) prealably heparinized (Heparin, Roche, Meylan, France). Blood was immediately transferred in a cold BD Vacutainer tube, centrifuged at 4,000 x *g* for 20 min at 4°C. Plasma was frozen and stored in

The steady state levels of OAT and cyclophilin A transcripts were estimated by semiquantitative polymerase chain reaction (PCR) and quantitative PCR (qPCR) as described

Total RNA was extracted from whole kidney by using Trizol® according to the supplier's procedure. Briefly, kidneys were mixed in the proportion of 150 mg tissue per 1 mL Trizol® at 4°C with a Ultra-Turrax T10 (VWR, Fontenay-sous-Bois, France). RNAs were extracted with chloroform, purified by isopropanol precipitation, and washed with 70% ethanol. RNA pellets were resuspended in sterilized water (Eurobio, Courtaboeuf, France) and stored

support a marked sexual dimorphism in the expression of OAT gene in the mouse kidney with three-fold higher levels in females than in males. A detailed study of the renal expression of OAT gene during the postnatal development of male and female mice revealed that puberty is the starter responsible for this sexual dimorphism (Levillain et al., 2007). In addition, the level of OAT protein has been inversely correlated with the plasma level of testosterone (Levillain et al., 2005).

The present study was designed to explore the mechanismes involved in the sexual dimorphism of the expression of OAT gene in the mouse kidney. We shall determine by *in vivo* studies at which level testosterone regulates the expression of OAT gene. To answer this question, male mice were subjected to orchidectomy and testosterone replacement. The renal expression of OAT gene was analyzed at the transcriptional, translational, and posttranslational levels. The possible involvement of the eukaryotic initiation factor eIF4-E in the control of OAT gene expression was analyzed. We also explored the delay required for testosterone to induce a decrease in the expression of OAT gene in orchidectomized mice. For this, a time-course study was performed over a period of 32 hrs following a single injection of testosterone to castrated male mice. Finally, to explain the physiological role of testosterone on the expression of OAT specifically in the mouse kidney, we searched to identify androgen response elements (ARE) in the promoter of the murine OAT gene.

#### **2. Material and methods**

#### **2.1 Animals and treatment**

Eight- to nine-week-old adult male (35-40 g body weight) OF-1 Swiss (IOPS Caw) mice, from either Charles River Laboratories (L'Arbresle-sur-Orge, France) or Janvier (Le Genestsaint-Isle, France) had free access to tap water and standard food (2018 Teklad Global 18% Protein Rodent Diet, Harlan, Gannat, France). Animals were housed in a controlled environment maintained at 21 ± 1°C with a 12-h light, 12-h dark cycle, lights on at 0600 h.

Twenty-four thirty-day-old male mice were subdivided into four groups of six mice: nonoperated (group I, control), sham-operated (group II) and two groups of orchidectomized mice. Eleven days later (*i.e.* 41 days after birth), mice of groups I, II, and III were sacrified, whereas mice of group IV were sacrified seven days latter (*i.e.* 18 days after orchidectomy).

Twenty-four thirty-day-old male mice were subdivided into four groups of six mice: shamoperated (group V, control), 11-*day* orchidectomized (group VI), 11-*day* orchidectomized treated with sesame oil (group VII), and 11-*day* orchidectomized treated with testosterone + sesame oil (group VIII). Mice subjected to oil or testosterone treatment were injected subcutaneously with 150 µL vehicule or testosterone propionate (3.1 mg/mL in sesame oil, i.e. approximately 15 µg/g BW or 0.55 mg per mouse). All mice were sacrified forty-eight hrs after the treatment.

Twenty thirty-day-old male mice were subjected to orchidectomy (11-*day*) and subdivided into five groups of four mice: 11-*day* orchidectomized (group IX, control) and four groups of mice treated with testosterone as described above (0.55 mg per mouse). Mice were sacrified 8 hrs (group X), 24 hrs (group XI), 28 hrs (group XII), or 32 hrs (group XIII) after the treatment (Fig. 1). For these three protocols, half of the mice were purchased from Charles River Laboratories and the other from Janvier. Mice were equally distributed in the different experimental groups. Orchidectomy was carried out by the suppliers. Mice were anesthetized (ip) using 0.1 mL/30 g BW pentobarbital sodium (Nembutal 6%, Clin Midy, Paris, France) diluted 1:2 in 0.9% NaCl solution.

support a marked sexual dimorphism in the expression of OAT gene in the mouse kidney with three-fold higher levels in females than in males. A detailed study of the renal expression of OAT gene during the postnatal development of male and female mice revealed that puberty is the starter responsible for this sexual dimorphism (Levillain et al., 2007). In addition, the level of OAT protein has been inversely correlated with the plasma

The present study was designed to explore the mechanismes involved in the sexual dimorphism of the expression of OAT gene in the mouse kidney. We shall determine by *in vivo* studies at which level testosterone regulates the expression of OAT gene. To answer this question, male mice were subjected to orchidectomy and testosterone replacement. The renal expression of OAT gene was analyzed at the transcriptional, translational, and posttranslational levels. The possible involvement of the eukaryotic initiation factor eIF4-E in the control of OAT gene expression was analyzed. We also explored the delay required for testosterone to induce a decrease in the expression of OAT gene in orchidectomized mice. For this, a time-course study was performed over a period of 32 hrs following a single injection of testosterone to castrated male mice. Finally, to explain the physiological role of testosterone on the expression of OAT specifically in the mouse kidney, we searched to identify androgen

Eight- to nine-week-old adult male (35-40 g body weight) OF-1 Swiss (IOPS Caw) mice, from either Charles River Laboratories (L'Arbresle-sur-Orge, France) or Janvier (Le Genestsaint-Isle, France) had free access to tap water and standard food (2018 Teklad Global 18% Protein Rodent Diet, Harlan, Gannat, France). Animals were housed in a controlled environment maintained at 21 ± 1°C with a 12-h light, 12-h dark cycle, lights on at 0600 h. Twenty-four thirty-day-old male mice were subdivided into four groups of six mice: nonoperated (group I, control), sham-operated (group II) and two groups of orchidectomized mice. Eleven days later (*i.e.* 41 days after birth), mice of groups I, II, and III were sacrified, whereas mice of group IV were sacrified seven days latter (*i.e.* 18 days after orchidectomy). Twenty-four thirty-day-old male mice were subdivided into four groups of six mice: shamoperated (group V, control), 11-*day* orchidectomized (group VI), 11-*day* orchidectomized treated with sesame oil (group VII), and 11-*day* orchidectomized treated with testosterone + sesame oil (group VIII). Mice subjected to oil or testosterone treatment were injected subcutaneously with 150 µL vehicule or testosterone propionate (3.1 mg/mL in sesame oil, i.e. approximately 15 µg/g BW or 0.55 mg per mouse). All mice were sacrified forty-eight

Twenty thirty-day-old male mice were subjected to orchidectomy (11-*day*) and subdivided into five groups of four mice: 11-*day* orchidectomized (group IX, control) and four groups of mice treated with testosterone as described above (0.55 mg per mouse). Mice were sacrified 8 hrs (group X), 24 hrs (group XI), 28 hrs (group XII), or 32 hrs (group XIII) after the treatment (Fig. 1). For these three protocols, half of the mice were purchased from Charles River Laboratories and the other from Janvier. Mice were equally distributed in the different experimental groups. Orchidectomy was carried out by the suppliers. Mice were anesthetized (ip) using 0.1 mL/30 g BW pentobarbital sodium (Nembutal 6%, Clin Midy,

level of testosterone (Levillain et al., 2005).

**2. Material and methods 2.1 Animals and treatment** 

hrs after the treatment.

Paris, France) diluted 1:2 in 0.9% NaCl solution.

response elements (ARE) in the promoter of the murine OAT gene.

Fig. 1. Schema of the time-course study of testosterone effect on the expression of OAT gene.

Animal care complied with French regulations for the protection of animals used for experimental and other scientific purposes and with European Community regulations (Council of Europe N° 123, Strasbourg, 1985). The author (O. Levillain) is authorized by the "Direction Départementale des Services Vétérinaires" (authorization no. 69-33 and 69266391) and the local Animal Care Committee to use animals for these experiments (authorization no. 299 revised and no. BH 2009-15).
