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The effect of hypothyroidism, induced by 6-n-propyl-2-thiouracil (PTU) administration to rats, on the retinoylation reaction and oxidative status was investigated in rat-testes mitochondria. In hypothyroid mitochondria, when compared to euthyroid controls, we found a noticeable increase in the amount of all-trans-retinoic acid (atRA) bound to mitochondrial proteins by an acylation process (34.2 +/- 1.9 pmoles atRA/mg protein/360 min and 22.2 +/- 1.7 pmoles atRA/mg protein/360 min, respectively). This increase, which was time- and temperature-dependent, was accompanied by a strong reduction in the cardiolipin (CL) amount in the mitochondrial membranes of hypothyroid (2.6 +/- 0.2%) as compared to euthyroid rats (4.5 +/- 0.5%) Conversely, a decreased retinoylation reaction was observed when CL liposomes were added to mitochondria or mitoplasts from both euthyroid and hypothyroid rats, thus confirming a role of CL in the retinoylation process. In mitochondria from the latter animals an increase of the level of oxidized CL occurred. The ATP level, which was reduced in hypothyroid mitochondria (27.3 +/- 4.1 pmoles ATP/mg protein versus 67.1 +/- 8.3 pmoles ATP/mg protein of euthyroid animals), was surprisingly increased in mitochondria by the retinoylation reaction in the presence of 100 nM atRA (481.5 +/- 19.3 pmoles ATP/mg protein of hypothyroid animals versus 84.7 +/- 7.7 pmoles ATP/mg protein of euthyroid animals). Overall, in hypothyroid rat-testes mitochondria the increase in retinoylation activity correlates with a significant depletion of the CL level, due to a peroxidation of this lipid. In addition, an enhanced production of reactive oxygen species was observed.

This study was undertaken to better understand the physiological role of the retinoylation process in steroidogenic tissues. In adrenal gland mitochondria, the retinoylation extent was found equal to that of testes mitochondria but without ATP in the incubation buffer. We pointed out that the endogenous mitochondrial ATP in adrenal glands is much higher than in testes, about 1.3 x 10(-2) M and 5.2 x 10(-8) M, respectively. In addition, less CoASH is required for the maximal acylation activity of the retinoyl moiety to protein(s) compared to testes. The fatty acid analysis revealed a different composition of mitochondrial membranes of these two tissues. Among the different values of fatty acids, it is important to note that adrenal glands contain a much higher amount of C18:0 and a much lower amount of C22:5 omega6 and C22:6 omega3 than testes in the mitochondrial membranes. In addition, there were also differences in arachidonic acid (ARA, C20:4 omega6) content between adrenal glands and testes mitochondria. These different values in the fatty acids composition should explain the different extent of the retinoylation process between the two organs.

In addition to playing a fundamental role in diverse processes, such as vision, growth and differentiation, vitamin A and its main biologically active derivative, retinoic acid (RA), are clearly involved in the regulation of testicular functions. The present study was undertaken to examine the direct effect of RA treatment on Leydig (TM-3) cells. TM-3 cells were cultured and treated with varying concentrations of RA for 24h. High doses of RA (1-20microM) induced a decrease in cell vitality and an increase in lipid peroxidation. RA treatment also induced a corresponding increase in apoptosis in the same cells in a dose-dependent manner. Apoptosis proceeded via the mitochondrial dependent pathway, as demonstrated by the release of cytochrome c, caspase-3 enzymatic activation and DNA fragmentation. Conversely, at physiological doses (0.1-500nM) RA did not increase lipid peroxidation or cell death and resulted in an increase of antioxidant enzyme activity.

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. The effects of RA are thought to be mediated by the nuclear retinoid receptors; however, not all the effects of RA can be explained by the nuclear receptor pathways. Indeed, retinoylation is another mechanism of action elicited by RA. In growing TM-3 Leydig cell cultures, the extent of retinoylation depends in a saturable manner on the initial concentration of 3H-RA, time and cell number. In addition, dose-response curves for RA-induced testosterone production and retinoylation are concomitant and exhibit a positive correlation. In the present study we demonstrate that RA is able to influence a retinoylation reaction on protein(s) probably involved on steroidogenesis.

Ferulic acid plays a chemopreventive role in cancer by inducing tumor cells apoptosis. As mitochondria play a key role in the induction of apoptosis in many cells types, here we investigate the mitochondrial permeability transition (MPT) and the release of cytochrome c induced by ferulic acid and its esters in rat testes mitochondria, in TM-3 and MLTC-1 cells. While ferulic acid, but not its esters, induced MPT and cytochrome c release in rat testes isolated mitochondria, in TM-3 cells we found that both ferulic acid and its esters induced cytochrome c release from mitochondria in a dose-dependent manner, suggesting a potential target of these compounds in the induction of cell apoptosis. The apoptosis induced by ferulic acid is therefore associated with the mitochondrial pathway involving cytochrome c release and caspase-3 activation.

All-trans-retinoic acid (atRA) is incorporated covalently into proteins of rat testes mitochondria. In this study, the effect of three diets with different fatty acid composition on the retinoylation of proteins of rat testes mitochondria has been investigated. Different groups of rats were fed on a basal diet supplemented with 15% of either coconut oil (CO), olive oil (OO) or fish oil (FO). We found that, when compared with CO, the binding of retinoic acid was decreased in FO- and OO-fed rats. Mitochondrial phospholipids composition was differently influenced by dietary treatments; minor changes were observed in fatty acid composition of phospholipids. Few differences were observed in the Arrhenius plots among the three groups of rats. Kinetic analysis revealed a decrease in the V (max) value in FO- and OO- as compared with CO-fed rats. No difference among the three groups were observed in the K ( M ) value. The retinoylation reaction was inhibited by 13-cis-RA and 9-cis-RA.

The covalent incorporation of [(3)H]all-trans-retinoic acid into proteins has been studied in tumoural Leydig (MLTC-1) cells. The maximum retinoylation activity of MLTC-1 cell proteins was 710+/-29 mean+/-SD) fmoles/8 x 10(4) cells at 37 degrees C. About 90% of [(3)H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65--75% after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC(50) values of 0.9 microM and 0.65 microM, respectively. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (250 microM); but there was an increase of the binding activity of about 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250 microM lauric acid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100 nM [(3)H]RA involved first an increase at 100 nM RA and then a decrease of retinoylation activity between 200 and 600 nM RA. After cycloheximide treatment of the tumoural Leydig cells the binding activity of [(3)H]RA was about the same as that in the control, suggesting that the bond occurred on proteins in pre-existing cells., ((Mol Cell Biochem 276: 55-60, 2005).)

The covalent incorporation of [(3)H]all-trans-retinoic acid into proteins has been studied in Leydig (TM-3) cells. The maximum retinoylation activity of Leydig cells proteins was 570+/- 27 fmoles/8 x 10(4) cells at 37( composite function)C. About 95% of [(3)H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion or after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation process was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC(50) values of 0.6 and 1.2 microM respectively. Dibutyryl-cAMP and forskolin increased the retinoylation activity by 75 and 81% at 500 and 25 microM respectively. Also hCG increased the retinoylation binding activity of 110% at 250 ng/mL. After cycloheximide treatment of the Leydig cells the binding activity of [(3)H]RA was about the same that in the control, suggesting that the bond occurs on proteins in pre-existing cells. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (500 microM); on the contrary, there was an increase of the binding activity of about 60 and 50% respectively.

Retinoylation (retinoic acid acylation), a posttranslational modification of proteins occurring in a variety of eukariotic cell lines both in vivo and in vitro, was studied in rat testes mitochondria. all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins of rat testes mitochondria. The maximum retinoylation activity of rat testes mitochondrial proteins was 21.6 pmoles mg protein(-1) 90 min(-1) at 37 degrees C. The activation energy was 44 kJ mol(-1) from 5 to 37 degrees C. The retinoylation activity had a pH optimum of 7.5. The retinoylation process was specific for the presence of ATP, ADP, and GTP (even if only 30% of the control). The half saturation constant (Km) was 0.69 microM for all-trans-retinoic acid, while the inhibition constant (Ki) was 1.5 microM for 13-cis-retinoic acid. Retinoylation was not inhibited by high concentrations of myristic acid (MA) and palmitic acid (PA), indicating that retinoylation and acylation reactions involved different rat testes mitochondrial proteins. The ATP or CoASH saturation curves of retinoylation reaction showed sigmoidal behavior with apparent half saturation constants (K0.5) of 6.5 mM ATP and 40.6 microM CoASH. On SDS-gel electrophoresis, the hydroxylapaptite/celite eluate showed various protein bands between 25 and 80 kDa. This retinoylated protein was purified 17-fold with respect to the mitochondrial extract.