Extra-virgin olive oil ameliorates high-fat diet-induced seminal and testicular disorders by modulating the cholesterol pathway.

BACKGROUND: Rabbits are sensitive to dietary cholesterol and rapidly develop hypercholesterolemia, leading to prominent subfertility. Sterol regulatory element-binding protein isoform 2 drives the intracellular cholesterol pathway in many tissues, including the testicles. Its abnormal regulation could be the mainly responsible for the failure of suppressing cholesterol synthesis in a cholesterol-enriched environment, ultimately leading to testicular and seminal alterations. However, extra-virgin olive oil consumption has beneficial properties that promote lowering of cholesterol levels, including the recovery of seminal parameters altered under a high-fat diet. OBJECTIVES: Our goal was to investigate the effects of high-fat diet supplementation with extra-virgin olive oil at the molecular level on rabbit testes, by analyzing sterol regulatory element-binding protein isoform 2 protein and its corresponding downstream effectors. MATERIALS AND METHODS: During 12 months, male rabbits were fed a control diet, high-fat diet, or 6-month high-fat diet followed by 6-month high-fat diet plus extra-virgin olive oil. Serum lipids, testosterone levels, bodyweight, and seminal parameters were tested. The mRNA and protein levels of sterol regulatory element-binding protein isoform 2, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, and low-density lipoprotein receptor were determined by semi-quantitative polymerase chain reaction and Western blotting techniques. The expression pattern of sterol regulatory element-binding protein isoform 2 protein in the rabbit testicles was studied by indirect immunofluorescence. In addition, testicular cholesterol was detected and quantified by filipin staining and gas chromatography. RESULTS: The data showed that the addition of extra-virgin olive oil to high-fat diet reduced testicular cholesterol levels and recovered the expression of sterol regulatory element-binding protein isoform 2, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, and low-density lipoprotein receptor initially altered by the high-fat diet. DISCUSSION AND CONCLUSIONS: The combination of high-fat diet with extra-virgin olive oil encourages testicular recovery by modifying the expression of the enzymes related to intracellular cholesterol management.

Consumption of oil macerated with garlic produces renovascular protective effects in adult apolipoprotein E-deficient mice.

Oxidative stress and chronic inflammatory conditions contribute as key determinants in the development of vascular and renal diseases. Organosulfur compounds (OSCs) of oil macerated with garlic (OMG) are promising phytochemicals which could protect us from hyper-inflammation and oxidative stress-induced organ damage. The present work evaluated the effect of OMG intake in apolipoprotein E-knockout (ApoE-KO) mice. Adult female ApoE-KO mice were randomly divided into three groups and fed with control chow, oil-supplemented diet and OMG-supplemented diet. After 8 weeks, the animals were euthanized and blood, aorta, kidneys, liver and abdominal adipose tissues were obtained for further analysis. Biochemical parameters were measured in plasma, lipid peroxidation as malondialdehyde (MDA) levels was determined in the adipose tissue, oil red O was used to stain atherosclerotic lesions, and histological and ultrastructural analyses of the kidneys were performed. Renal expression levels of Tumor Necrosis Factor α (TNF-α), Interleukin-6 (IL-6) and Wilms' Tumor Protein (WT-1) were determined by western blotting and the co-immunoprecipitation assay (p53/WT-1). Also, transmission electron microscopy for studying the expression of mitofusin 2 (Mfn-2) was used to assess mitochondrial damage. The results showed that long-term moderate intake of OMG improved serum triglyceride levels, diminished the atheroma plaque area, and reduced lipid peroxidation. Furthermore, we found a decrease in oxidative and inflammatory markers, less apoptosis and reduced WT-1 expression in the kidneys. Also, OMG increased p53/WT-1 protein interactions and reduced mitochondrial damage. Our findings suggest that OMG intake would produce anti-atherosclerotic, antifibrotic, anti-inflammatory and antiapoptotic effects in adult ApoE-KO mice, conferring significant renovascular protective actions in a mechanism mediated, at least in part, by WT-1.

Thiols of flagellar proteins are essential for progressive motility in human spermatozoa.

Male infertility is a disorder of the reproductive system defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse. The presence of low-motile or immotile spermatozoa is one of many causes of infertility; however, this observation provides little or no information regarding the pathogenesis of the malfunction. Good sperm motility depends on correct assembly of the sperm tail in the testis and efficient maturation during epididymal transit. Thiols of flagellar proteins, such as outer dense fibre protein 1 (ODF1), are oxidised to form disulfides during epididymal transit and the spermatozoa become motile. This study was designed to determine how oxidative changes in protein thiol status affect progressive motility in human spermatozoa. Monobromobimane (mBBr) was used as a specific thiol marker and disruptor of sperm progressive motility. When mBBr was blocked by dithiothreitol it did not promote motility changes. The analysis of mBBr-treated spermatozoa revealed a reduction of progressive motility and an increased number of spermatozoa with non-progressive motility without affecting ATP production. Laser confocal microscopy and western blot analysis showed that one of the mBBr-positive proteins reacted with an antibody to ODF1. Monobromobimane fluorescence intensity of the sperm tail was lower in normozoospermic than asthenozoospermic men, suggesting that thiol oxidation in spermatozoa of asthenozoospermic men is incomplete. Our findings indicate that mBBr affects the thiol status of ODF1 in human spermatozoa and interferes with progressive motility.

Effects of chronic exposure to cadmium on prostate lipids and morphology.

Cadmium is an environmental toxic metal implicated in human prostate carcinogenesis. The mechanism of its toxicity is not fully understood. Previously, we showed that cadmium exposure induces oxidative stress, especially lipid peroxidation. This study evaluates the effect of chronic exposure to 0.886 mM of cadmium (Cd) per liter in the drinking water on prostate lipid content and metabolism in Wistar rats. We determined the lipid profile and measured the expression of lipogenic enzymes: FAS, GPAT, LPL, DGAT-1, DGAT-2, ACO, CPT-1 and CT, and of certain factors involved in lipid regulation and fatty acid transporters: FAT/CD36, E-FABP, SREBP-2, PPAR-gamma and PPAR-alpha by RT-PCR. Ultrastructure was analyzed by electron microscopy and, as prostate is an androgen controlled gland, AR expression was measured by RT-PCR and Western blot. Cd altered the prostatic lipid profile. Triglycerides (TG) and esterified cholesterol (EC) decreased, free cholesterol (FC) and phospholipids (PL) increased and total cholesterol (TC) did not change. FAS, MDH and IDH activities did not vary but G6PDH decreased significantly in Cd group. Regarding TG synthesis, DGAT-1 decreased while GPAT increased and FAS, LPL and DGAT-2 remained unchanged. Regarding beta oxidation, CPT-1 increased while ACO expression decreased in Cd group. In the PL pathway, CT expression was increased. All these results would justify the decrease of TG in Cd group when compared to control. In the cholesterol metabolic pathway, HMGCoAR and SREBP-2 increased. PPAR-alpha increased but PPAR-gamma did not change. Regarding fatty acid transporters, FAT/CD36 decreased, while E-FABP increased. AR mRNA and protein expression decreased. Ultrastructural analysis showed a decrease in lipid droplets and signs of cellular damage in the Cd group. Cadmium exposure induces important changes in prostatic lipid profile and metabolism, confirmed by the morphology analyses, which also showed signs of cellular damage. These results could be important to further understanding the complex mechanism of cadmium toxicity in prostate and in the development of better treatments for people and animals exposed to the heavy metal.

Simultaneous activation of PLA2 and PLC are required to promote acrosomal reaction stimulated by progesterone via G-proteins.

The acrosome reaction (AR) is a special exocytotic process promoted by signal transduction pathways studied in many laboratories. Progesterone (P4) is one of the trigger molecules proposed. Upon the binding of P4 to its receptor, several molecules could be activated, including G-proteins, phospholipase A(2) (PLA(2)), and phospholipase C (PLC). The role of these molecules was analyzed in this study using the Chlortetracycline (CTC) protocol to detect and quantify the AR. Incubation of capacitated sperm cells with GTPgammas (GTPgammas, a mimetic of G-protein activation), arachidonic acid (AA, product of PLA(2) action), or phorbol ester (PMA, an activator of PLC) for 15 min increased the AR to a similar percentage as P4. Conversely, a decrease in the AR was detected when sperm cells were incubated with P4 after preincubation with: GDPbetaS (GDP, an inhibitor of G-protein activation), ONO RS-82 (ONO, an inhibitor of PLA(2)), or neomycin (Neo, an inhibitor of PLC) for 15 min. To analyze the activation sequence of G proteins, PLA(2), and PLC combinations of these mimetic/inhibitors were used during successive incubation periods. Inhibition promoted by GDP, ONO, and Neo were overcome by 15-min incubation with GTPgammas, AA, or PMA, respectively. But GTPgammas or P4 did not reverse the inhibition due to incubation with Neo and ONO. Interestingly, this dual inhibition was reverted by another 15-min incubation with AA or PMA. Results presented here could indicate that the AR triggered by P4 is driven by activation of G-proteins, that in turn activate PLA(2) and PLC simultaneously, that finally promote acrosomal exocytosis.