Search

Diabetes mellitus (DM) is a metabolic disease that is rapidly increasing and has become a major public health problem. Type 2 DM (T2DM) is the most common type, accounting for up to 90-95% of the new diagnosed DM cases. The brain is very susceptible to glucose fluctuations and hyperglycemia-induced oxidative stress (OS). It is well known that DM and the risk of developing neurodegenerative diseases are associated. Tea, Camellia sinensis L., is one of the most consumed beverages. It contains several phytochemicals, such as polyphenols, methylxanthines (mainly caffeine) and L-theanine that are often reported to be responsible for tea's health benefits, including in brain. Tea phytochemicals have been reported to be responsible for tea's significant antidiabetic and neuroprotective properties and antioxidant potential. Epidemiological studies have shown that regular consumption of tea has positive effects on DM-caused complications and protects the brain against oxidative damage, contributing to an improvement of the cognitive function. Among the several reported benefits of tea consumption, those related with neurodegenerative diseases are of great interest. Herein, we discuss the potential beneficial effects of tea consumption and tea phytochemicals on DM and how their action can counteract the severe brain damage induced by this disease.

The fruits of Solanum lycocarpum, known as wolf-fruit, are used in folk medicine, and because of that we have evaluated both the genotoxic potential of its glycoalkaloidic extract (SL) and its influence on the genotoxicity induced by methyl methanesulfonate. Furthermore, the potential blocking effect of SL intake in the initial stage of colon carcinogenesis in Wistar rats was investigated in a short-term (4-week) bioassay using aberrant crypt foci (ACF) as biomarker. The genotoxic potential was evaluated using the Swiss mice peripheral blood micronucleus test. The animals were treated with different doses of SL (15, 30 and 60 mg/kg b.w.) for 14 days, and the peripheral blood samples were collected at 48 h, 7 days and 14 days after starting the treatment. For antigenotoxicity assessment, MMS was administered on the 14th day, and after 24 h the harvesting of bone marrow and liver cells was performed, for the micronucleus and comet assays, respectively. In the ACF assay, male Wistar rats were given four subcutaneous injections of the carcinogen 1,2-dimethylhydrazine (DMH, 40 mg/kg b.w.), twice a week, during two weeks to induce ACF. The treatment with SL (15, 30 and 60 mg/kg b.w.) was given for four weeks during and after carcinogen treatment to investigate the potential beneficial effects of SL on DMH-induced ACF. The results demonstrated that SL was not genotoxic in the mouse micronucleus test. In animals treated with SL and MMS, the frequencies of micronucleus and extensions of DNA damage were significantly reduced in comparison with the animals receiving only MMS. Regarding the ACF assay, SL significantly reduced the frequency of ACF induced by DMH.

Melatonin co-operates with insulin in the regulation of glucose homeostasis. Within the testis, glucose metabolism in the somatic Sertoli cells (SCs) is pivotal for spermatogenesis. Since the effects of melatonin on male reproductive physiology remain largely unknown, we hypothesized that melatonin may affect spermatogenesis by modulating SC metabolism, interacting with insulin. To test our hypothesis, rat SCs were maintained in culture for 24 h in the presence of insulin, melatonin or both and metabolite production/consumption was determined by proton nuclear magnetic resonance ((1)H-NMR). Protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were determined by western blot. LDH activity was also assessed. SCs treated with melatonin showed an increase in glucose consumption via modulation of GLUT1 levels, but decreased LDH protein expression and activity, which resulted in lower lactate production. Moreover, SCs exposed to melatonin produced and accumulated less acetate than insulin-exposed cells. The combined treatment (insulin plus melatonin) increased acetate production by SCs, but intracellular acetate content remained lower than in insulin exposed cells. Finally, the intracellular redox state, as reflected by intracellular lactate/alanine ratio, was maintained at control levels in SCs by melatonin exposure (i.e. melatonin, alone or with insulin, increased the lactate/alanine ratio versus cells treated with insulin). Furthermore, SCs exposed to insulin plus melatonin produced more lactate and maintained the protein levels of some glycolysis-related enzymes and transporters at control levels. These findings illustrate that melatonin regulates SCs metabolism, and thus may affect spermatogenesis. Since lactate produced by SCs provides nutritional support and has an anti-apoptotic effect in developing germ cells, melatonin supplementation may be an effective therapy for diabetic male individuals facing subfertility/infertility., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Individuals with diabetes mellitus (DM) present marked reduction in sperm quality and higher DNA damage in spermatozoa, evidencing that this metabolic disorder impairs male fertility. These effects are related to defective testicular metabolic pathways and signaling, resulting in altered sperm metabolism. Spermatozoa metabolize several substrates to ensure energy supplies and any alteration in this feature compromise sperm quality. For ATP production, spermatozoa require substrate availability and the involvement of specific hexose membrane carriers. DM is known to modulate the spermatozoa substrate consumption and/or production due to altered glycolytic behavior. In fact, glucose uptake and metabolism is highly deregulated in diabetic individuals. Herein, we present an overview of the implications of DM in sperm glucose uptake and metabolism. The understanding of these processes is essential to identify key mechanisms associated with DM-related male (in)fertility. Moreover, it may contribute to the development of therapeutics to counteract the dysfunction induced by DM in sperm metabolism., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Salvia officinalis (sage) is a perennial woody subshrub native to the Mediterranean region that is commonly used as a condiment and as an anti-inflammatory, antioxidant and antimicrobial agent due to its biological activities. Manool is the most abundant micro-metabolite found in Salvia officinalis essential oils and extracts. We therefore decided to evaluate the cytotoxic, genotoxic and antigenotoxic potential of manool in Chinese hamster lung fibroblasts (V79) and human hepatoma cells (HepG2). Cytotoxicity was assessed by the colony-forming assay in V79 cells and toxic effects were observed at concentrations of up to 8.0 μg/mL. The micronucleus test was used to evaluate the genotoxicity and antigenotoxicity of manool in V79 and HepG2 cells at concentrations of 0.5-6.0 μg/mL and 0.5-8.0 μg/mL, respectively. For evaluation of antigenotoxicity, the concentrations of manool were combined with methyl methanesulfonate (MMS, 44 μg/mL). The results showed a significant increase in the frequency of micronuclei in cultures of both cell lines treated with the highest concentration tested, demonstrating a genotoxic effect. On the other hand, manool exhibited a protective effect against chromosome damage induced by MMS in HepG2 cells, but not in V79 cells. These data suggest that some manool metabolite may be responsible for the antigenotoxic effect observed in HepG2 cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mitochondria have a crucial role in the supply of energy to the brain. Mitochondrial alterations can lead to detrimental consequences on the function of brain cells and are thought to have a pivotal role in the pathogenesis of several neurologic disorders. This study was aimed to evaluate mitochondrial function, fusion-fission and biogenesis and autophagy in brain cortex of 6-month-old Goto-Kakizaki (GK) rats, an animal model of nonobese type 2 diabetes (T2D). No statistically significant alterations were observed in mitochondrial respiratory chain and oxidative phosphorylation system. A significant decrease in the protein levels of OPA1, a protein that facilitates mitochondrial fusion, was observed in brain cortex of GK rats. Furthermore, a significant decrease in the protein levels of LC3-II and a significant increase in protein levels of mTOR phosphorylated at serine residue 2448 were observed in GK rats suggesting a suppression of autophagy in diabetic brain cortex. No significant alterations were observed in the parameters related to mitochondrial biogenesis. Altogether, these results demonstrate that during the early stages of T2D, brain mitochondrial function is maintained in part due to a delicate balance between mitochondrial fusion-fission and biogenesis and autophagy. However, future studies are warranted to evaluate the role of mitochondrial quality control pathways in late stages of T2D.

Purpose: Many health benefits have been attributed to tea (Camellia sinensis (L.)), and tea infusions are used as dietary agent and included in food supplements. Herein, we report the effect of a white tea (WTEA) extract in Sertoli cell (SC) metabolism. The SC is responsible for the nutritional support of the developing germ cells., Methods: An aqueous WTEA extract was prepared and analyzed by (1)H-NMR. Rat SCs were cultured with or without the WTEA extract. mRNA and protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were determined by qPCR and western blot. LDH activity was assessed and metabolite production/consumption determined by (1)H-NMR., Results: WTEA-exposed SCs presented decreased protein and mRNA levels of GLUT1 and decreased glucose uptake. However, intracellular LDH activity was increased and SC lactate production was stimulated by the presence of the WTEA extract. Interestingly, alanine production was also found to be stimulated in WTEA extract-exposed SCs., Conclusion: WTEA extract altered the glycolytic profile of cultured SCs, stimulating lactate production. Since lactate is used as metabolic substrate and has an anti-apoptotic effect in the developing germ cells, the supplementation with WTEA extract may be advantageous to improve male reproductive health.

Men with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are usually subfertile/infertile. Besides playing a role in Cl(-)/HCO3(-) transport, it has been proposed that CFTR interacts with water membrane transport systems, particularly aquaporins, to control seminiferous tubular secretion, which is regulated by the somatic Sertoli cells (SCs). As aquaporin-9 (AQP9) is highly expressed throughout the male reproductive tract, we hypothesized that it is also present in rat SCs and that it physically interacts with CFTR. To test this hypothesis, primary cultures of rat SCs were established, and expression of CFTR and AQP9 was assessed by RT-polymerase chain reactions (mRNA) and Western blot analysis (protein). A coimmunoprecipitation assay was used to evaluate the physical interaction between CFTR and AQP9. Our results show that CFTR and AQP9 are expressed in rat SCs. We were also able to detect a molecular interaction between CFTR and AQP9 in rat SCs. This is the first report describing the presence of AQP9, and its interaction with CFTR, in rat SCs. Moreover, our results provide evidence that CFTR is involved in water homeostasis of the seminiferous tubular secretion. These mechanisms may open new insights on therapeutic targets to counteract subfertility/infertility in men with cystic fibrosis and mutations in the CFTR gene., (© 2014 International Union of Biochemistry and Molecular Biology.)

Background: Regucalcin (RGN) is a calcium (Ca(2+) )-binding protein underexpressed in prostate adenocarcinoma comparatively to non-neoplastic prostate or benign prostate hyperplasia cases. Moreover, RGN expression is negatively associated with the cellular differentiation of prostate adenocarcinoma, suggesting that loss of RGN may be associated with tumor onset and progression. However, the RGN actions over the control of prostate cell growth have not been investigated., Methods: Androgens are implicated in the promotion of prostate cell proliferation, thus we studied the in vivo effect of androgens on RGN expression in rat prostate. The role of RGN modulating cell proliferation and apoptotic pathways in rat prostate was investigated using transgenic animals (Tg-RGN) overexpressing the protein., Results: In vivo stimulation with 5α-dihydrotestosterone (DHT) down-regulated RGN expression in rat prostate. Cell proliferation index and prostate weight were reduced in Tg-RGN, which was concomitant with altered expression of cell-cycle regulators. Tg-RGN presented diminished expression of the oncogene H-ras and increased expression of cell-cycle inhibitor p21. Levels of anti-apoptotic Bcl-2, as well as the Bcl-2/Bax protein ratio were increased in prostates overexpressing RGN. Both caspase-3 expression and enzyme activity were decreased in the prostates of Tg-RGN., Conclusions: Overexpression of RGN resulted in inhibition of cell proliferation and apoptotic pathways, which demonstrated its role maintaining prostate growth balance. Thus, deregulation of RGN expression may be an important event favoring the development of prostate cancer. Moreover, the DHT effect down-regulating RGN expression in rat prostate highlighted for the importance of this protein in prostatic physiology., (© 2014 Wiley Periodicals, Inc.)

Bivalve molluscs, due to their sedentary mode of life and filter-feeding behavior, are very susceptible to pollutant bioaccumulation and used as sentinel organisms in the assessment of environment pollution. Herein we aimed to determine the in vivo, ex vivo and in vitro effects of 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, in Anodonta cygnea shell growth mechanisms. For that, we evaluated the effect of 2,4-D (100 μM) exposure on the transepithelial short-circuit current (Isc), potential (Vt) and conductance (Gt), as well as on OME ion transport systems and intracellular pH (pHi). In vivo exposure to 2,4-D caused an increase of 50% on the Isc generated by OME and ex vivo addition of that compound to the apical side of OME also induced an Isc increase. Furthermore, 2,4-D was able to cause a pHi increase in isolated cells of OME. Noteworthy, when 2,4-D was added following the exposure to specific inhibitors of several membrane transporters identified as responsible for pHi maintenance in these cells, no significant effect was observed on pHi except when the V-type ATPase inhibitor was used, indicating an overlap with the effect of 2,4-D. Thus, we concluded that 2,4-D is able of enhancing the activity of the V-ATPases present on the OME of A. cygnea and that this effect seems to be due to a direct stimulation of those H(+) transporters present on the apical portion of the membrane of OME cells, which are vital for shell maintenance and growth. This study allows us to better understand the molecular mechanisms behind 2,4-D toxicity and its deleterious effect in aquatic ecosystems, with particular emphasis on those involved in shell formation of bivalves., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The maintenance of pH homeostasis in the male reproductive tract is kept through the involvement of several mechanisms, among which is included the transmembranous movement of H(+) ions. Na(+)-H(+) exchangers (SLC9, solute carrier 9 family members) are among the membrane transporters known to participate in intracellular and extracellular pH regulation but also have important roles in salt and water absorption across epithelia and in the regulation of cell volume. The presence of several Na(+)-H(+) exchangers has been reported in the male reproductive tract. Their involvement in the processes that ensure the correct pursuance of the spermatogenetic event and spermatozoa maturation has been suggested. Indeed, the formation of mature spermatozoa is highly dependent on the maintenance of adequate ductal luminal milieu pH and ionic balance. Perturbations in these processes result in reduced male reproductive potential and consequently male subfertility and/or infertility. Thus, it is imperative to understand H(+) transport dynamics in order to identify and counteract possible alterations associated with reduced male fertility caused by pathological conditions. Herein, we will discuss the expression pattern and physiological roles of SLC9 family members in the cells of the male reproductive tract as well as the molecular basis of H(+) transport and its involvement in male reproductive potential., (© 2014 by the Society for the Study of Reproduction, Inc.)

Objective: To study the effect of estrogens regulating the testicular expression of stem cell factor (SCF) and c-kit., Design: Experimental study., Setting: University research center., Animal(s): Male Wistar rats., Intervention(s): Rat seminiferous tubules (SeT) cultured in the presence or absence of 17β-estradiol (E2)., Main Outcome Measure(s): Expression of SCF and c-kit as well as apoptotic factors, FasL, FasR, Bcl-2, and Bax analyzed via quantitative reverse transcription-polymerase and Western blot; enzymatic activity of apoptosis effector caspase-3 assessed by colorimetric assay; proliferation index in SeT epithelium determined via fluorescent immunohistochemistry of nuclear proliferation marker Ki67., Result(s): E2 (100 nM) induced a decrease in c-kit expression while increasing expression of SCF. Altered expression of the SCF/c-kit system relied on apoptosis of germ cells, as evidenced by the up-regulated expression of FasL/FasR, the increased ratio of proapoptotic/antiapoptotic proteins (Bax/Bcl-2), and the augmented activity of caspase-3. Decreased proliferation was also found in SeT in response to E2., Conclusion(s): A 100 nM dose of E2 unbalance the SCF/c-kit system, with a crucial impact on germ cell survival and thus male fertility. These findings contribute to our knowledge of the mechanisms underlying male idiopathic infertility associated with hyperestrogenism and open new perspectives on treatment targeting estrogen-signaling mechanisms., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The main purpose of this study was to examine whether streptozotocin (STZ)-induced type 1 diabetes (T1D) and insulin (INS) treatment affect mitochondrial function, fission/fusion and biogenesis, autophagy and tau protein phosphorylation in cerebral cortex from diabetic rats treated or not with INS. No significant alterations were observed in mitochondrial function as well as pyruvate levels, despite the significant increase in glucose levels observed in INS-treated diabetic rats. A significant increase in DRP1 protein phosphorylated at Ser616 residue was observed in the brain cortex of STZ rats. Also an increase in NRF2 protein levels and in the number of copies of mtDNA were observed in STZ diabetic rats, these alterations being normalized by INS. A slight decrease in LC3-II levels was observed in INS-treated rats when compared to STZ diabetic animals. An increase in tau protein phosphorylation at Ser396 residue was observed in STZ diabetic rats while INS treatment partially reversed that effect. Accordingly, a modest reduction in the activation of GSK3β and a significant increase in the activity of phosphatase 2A were found in INS-treated rats when compared to STZ diabetic animals. No significant alterations were observed in caspases 9 and 3 activity and synaptophysin and PSD95 levels. Altogether our results show that mitochondrial alterations induced by T1D seem to involve compensation mechanisms since no significant changes in mitochondrial function and synaptic integrity were observed in diabetic animals. In addition, INS treatment is able to normalize the alterations induced by T1D supporting the importance of INS signaling in the brain., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The role of estrogens in male reproductive physiology has been intensively studied over the last few years. Yet, the involvement of their specific receptors has long been a matter of debate. The selective testicular expression of the classic nuclear estrogen receptors (ERα and ERβ) argues in favor of ER-specific functions in the spermatogenic event. Recently, the existence of a G protein-coupled estrogen receptor (GPR30) mediating non-genomic effects of estrogens has also been described. However, little is known about the specific testicular expression pattern of GPR30, as well as on its participation in the control of male reproductive function. Herein, by means of immunohistochemical and molecular biology techniques (RT-PCR and Western blot), we aimed to present the first exhaustive evaluation of GPR30 expression in non-neoplastic human testicular cells. Indeed, we were able to demonstrate that GPR30 was expressed in human testicular tissue and that the staining pattern was consistent with its cytoplasmic localization. Additionally, by using cultured human Sertoli cells (SCs) and isolated haploid and diploid germ cells fractions, we confirmed that GPR30 is expressed in SCs and diploid germ cells but not in haploid germ cells. This specific expression pattern suggests a role for GPR30 in spermatogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Sertoli cells (SCs) form the blood-testis barrier (BTB) that controls the microenvironment where the germ cells develop. The cystic fibrosis transmembrane conductance regulator (CFTR) plays an essential role to male fertility and it was recently suggested that it may promote water transport. Interestingly, Aquaporin-4 (AQP4) is widely expressed in blood barriers, but was never identified in SCs. Herein we hypothesized that SCs express CFTR and AQP4 and that they can physically interact. Primary SCs cultures from 20-day-old rats were maintained and CFTR and AQP4 mRNA and protein expression was assessed by RT-PCR and Western blot, respectively. The possible physical interaction between CFTR and AQP4 was studied by co-immunoprecipitation. We were able to confirm the presence of CFTR at mRNA and protein level in cultured rat SCs. AQP4 mRNA analysis showed that cultured rat SCs express the transcript variant c of AQP4, which was followed by immunodetection of the correspondent protein. The co-immunoprecipitation experiments showed a direct interaction between AQP4 and CFTR in cultured rat SCs. Our results suggest that CFTR physically interacts with AQP4 in rat SCs evidencing a possible mechanism by which CFTR can control water transport through BTB. The full enlightenment of this particular relation between CFTR and AQP4 may point towards possible therapeutic targets to counteract male subfertility/infertility in men with Cystic Fibrosis and mutations in CFTR gene, which are known to impair spermatogenesis due to defective water transport., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This study evaluated the antiproliferative activity of the Brazilian green propolis and Baccharis dracunculifolia extracts and their major compounds artepillin C and baccharin in different tumor cell lines. The lowest IC50 values observed for Brazilian green propolis and B. dracunculifolia extracts were 41.0 ± 4.5 µg/mL for U343 and 44.9 ± 7.1 µg/mL for HepG2, respectively. Regarding artepillin C and baccharin, the lowest IC50 values were 20.1 ± 2.9 for U343 and 13.0 ± 1.5 µg/mL for B16F10, respectively. For the association of artepillin C plus baccharin, the lowest IC50 result was 35.2 ± 0.5 µg/mL for B16F10. Artepillin C and baccharin were more cytotoxic than both Brazilian green propolis and B. dracunculifolia extracts. No additive or synergistic effect was observed for the association of artepillin C plus baccharin., (Georg Thieme Verlag KG Stuttgart · New York.)

Recent evidence suggested the involvement of calcium-binding protein regucalcin (RGN) in testicular apoptosis. Herein, we investigated the role of RGN controlling apoptotic pathways in the testis by using a transgenic rat model overexpressing RGN (Tg-RGN). Seminiferous tubules (SeT) from Tg-RGN and their wild-type (Wt) counterparts were cultured ex vivo in presence or absence of apoptosis inducers thapsigargin (Thap, 10(-7) and 10(-6) m) and actinomycin D (Act D, 0.5 and 1 μg/mL). Expression levels of key regulators of apoptosis in SeT of Tg-RGN and Wt animals were determined by quantitative real-time PCR and Western blot analysis. Measurement of caspase-3 enzymatic activity was included as an end point of apoptosis. Tg-RGN SeT treated with 10(-6) m of Thap or 1 μg/mL of Act D showed a diminished enzymatic activity and gene transcription of caspase-3, along with increased mRNA and protein expression of antiapoptotic Bcl-2. Bcl-2/Bax (antiapoptotic/proapoptotic) protein ratio was also enhanced in these SeT. Although caspase-9 mRNA was increased in the SeT of Tg-RGN treated with Thap, no differences were observed at protein level, and no differences were also found on protein levels of apoptosis-inducing factor. mRNA expression of proapoptotic p53 and p21 was strongly decreased in Tg-RGN SeT treated with Thap (10(-6) m) or Act D (1 μg/mL). These findings demonstrated that RGN suppresses Thap- and Act D-induced apoptosis in SeT by modulating the expression and activity of key apoptotic and antiapoptotic factors. Moreover, results indicate that RGN overexpression protects germ cell from apoptosis induced by noxious stimuli, which could be a relevant mechanism for fertility preservation in situations of oncological treatments., (© 2014 American Society of Andrology and European Academy of Andrology.)

Pre-diabetes, a risk factor for type 2 diabetes development, leads to metabolic changes at testicular level. Peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) and Sirtuin 3 (Sirt3) are pivotal in mitochondrial function. We hypothesized that pre-diabetes disrupts testicular PGC-1α/Sirt3 axis, compromising testicular mitochondrial function. Using a high-energy-diet induced pre-diabetic rat model, we evaluated testicular levels of PGC-1α and its downstream targets, nuclear respiratory factors 1 (NRF-1) and 2 (NRF-2), mitochondrial transcription factor A (TFAM) and Sirt3. We also assessed mitochondrial DNA (mtDNA) content, mitochondrial function, energy levels and oxidative stress parameters. Protein levels were quantified by Western Blot, mtDNA content was determined by qPCR. Mitochondrial complex activity and oxidative stress parameters were spectrophotometrically evaluated. Adenine nucleotide levels, adenosine and its metabolites (inosine and hypoxanthine) were determined by reverse-phase HPLC. Pre-diabetic rats showed increased blood glucose levels and impaired glucose tolerance. Both testicular PGC-1α and Sirt3 levels were decreased. NRF-1, NRF-2 and TFAM were not altered. Testicular mtDNA content was decreased. Mitochondrial complex I activity was increased, whereas mitochondrial complex III activity was decreased. Adenylate energy charge was decreased in pre-diabetic rats, as were ATP and ADP levels. Conversely, AMP levels were increased, evidencing a decreased ATP/AMP ratio. Concerning to oxidative stress pre-diabetes decreased testicular antioxidant capacity and increased lipid and protein oxidation. In sum, pre-diabetes compromises testicular mitochondrial function by repressing PGC-1α/Sirt3 axis and mtDNA copy number, declining respiratory capacity and increasing oxidative stress. This study gives new insights into overall testicular bioenergetics at this prodromal stage of disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background and Purpose: Metformin is commonly used to treat type 2 diabetes (T2D). While new clinical applications have been ascribed to metformin, including treatment of anovulatory infertility, its effects on male reproduction have not been investigated. The Sertoli cell (SC) is crucial for germ cell development, exerting metabolic control of spermatogenesis, therefore, we investigated the effects of metformin on SC metabolism., Experimental Approach: Rat SCs were cultured in the absence and presence of metformin (5, 50 and 500 μM). mRNA and protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK 1), lactate dehydrogenase (LDH) and monocarboxylate transporter 4 (MCT4) were determined by quantitative PCR and Western blot respectively. LDH activity was assessed and metabolite production/consumption determined by (1) H-NMR., Key Results: Metformin (50 μM) decreased mRNA and protein levels of GLUT1, GLUT3, MCT4 and PFK 1 but did not affect LDH mRNA or protein levels. However, although glucose consumption was maintained in metformin-treated cells, LDH activity, lactate and alanine production were increased, indicating an enhanced glycolytic flux. No metabolic cytotoxicity was detected in SCs exposed to supra-pharmacological concentration of metformin., Conclusions and Implications: Our results indicate that metformin: (i) decreases mRNA and protein levels of glycolysis-related transporters in SCs but increases their activity; and (ii) stimulates alanine production, which induces antioxidant activity and maintains the NADH/NAD(+) equilibrium. The increased lactate in metformin-treated SCs provides nutritional support and has an anti-apoptotic effect in developing germ cells. Thus, metformin can be considered as a suitable antidiabetic drug for male patients of reproductive age with T2D., (© 2013 The British Pharmacological Society.)

Storage of sperm under refrigeration reduces its viability, due to oxidative unbalance. Unfermented teas present high levels of catechin derivatives, known to reduce oxidative stress. This study investigated the effect of white tea (WTEA) on epididymal spermatozoa survival at room temperature (RT), using green tea (GTEA) for comparative purposes. The chemical profiles of WTEA and GTEA aqueous extracts were evaluated by (1)H NMR. (-)-Epigallocatechin-3-gallate was the most abundant catechin, being twice as abundant in WTEA extract. The antioxidant power of storage media was evaluated. Spermatozoa antioxidant potential, lipid peroxidation, and viability were assessed. The media antioxidant potential increased the most with WTEA supplementation, which was concomitant with the highest increase in sperm antioxidant potential and lipid peroxidation decrease. WTEA supplementation restored spermatozoa viability to values similar to those obtained at collection time. These findings provide evidence that WTEA extract is an excellent media additive for RT sperm storage, to facilitate transport and avoid the deleterious effects of refrigeration.

Population growth in the last century has raised important social and economic questions. Thus, current methods of fertility control have been under debate for a long period. Birth rates are essentially dependent on several environmental and social factors but women, who are great users of contraceptives, play a major role. Regulation of male fertility has been widely studied in recent years with the aim of developing a new male contraceptive for further inclusion of men's choice in family planning. Based on the ancient people techniques to control the birth rates, natural products appeared as a promising source for the development of a male contraceptive. Over the years, many plants and their main constituents have been studied in the search for their antifertility properties. Interestingly, some antispermatogenic effects have been reported. Herein, we will discuss the antispermatogenic properties of some natural products. We propose to discuss specific targets and sites of action of the selected natural products. Despite the advances in this field in the last years, the molecular mechanisms by which natural products can control fertility, need to be disclosed to develop an effective, reversible and safe male contraceptive and avoid undesired toxicity in other organs. To date, no natural-based male contraceptive is available in the commercial market, mostly due to the difficulty in reversing the effects of these products in male fertility.

Bixin is a carotenoid found in the seeds of Bixa orellana L., a plant native to tropical America that is used in the food industry. The aim of this study was to investigate the effect of bixin on DNA damage and pre-neoplastic lesions induced by 1,2-dimethylhydrazine (DMH) in the liver and colon of Wistar rats. The animals received bixin at daily doses of 0.1, 1.0 and 10mg/kg body weight (bw) by gavage. For the assessment of DNA damage in hepatocytes and colon cells with the comet assay, the administration of bixin was for 7 days. The animals received a single subcutaneous injection of 25mg/kg bw of DMH, and were euthanized 4h later. For the evaluation of the frequency of aberrant crypt foci (ACF), the animals were treated with the different doses of bixin for 4 weeks. Four doses of 40mg/kg bw DMH, two doses in the first week and two doses in the second week, were administered and euthanasia occurred at 4 weeks after the beginning of treatment. Bixin reduced the frequency of DNA damage in hepatocytes at the highest two doses tested (1.0 and 10mg/kg bw). On the other hand, no differences in the frequency of DNA damage in colon cells were observed between animals treated with bixin plus DMH and those treated with DMH alone. In addition, the frequency of ACF did not differ significantly between the group treated with bixin plus DMH and the DMH group. The results suggest that bixin does not suppress the formation of ACF, indicating the absence of a protective effect against colon carcinogenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Natural products are some of the important sources of new anticancer drugs. The Brazilian flora is considered one of the most diverse in the word, although not many large-scale pharmacological and phytochemical studies have been conducted to date. With this in mind, in the present study we evaluated the antiproliferative activity of Solanum lycocarpum fruit glycoalkaloid extract (SL) and its major compounds, solamargine (SM) and solasonine (SS), against different tumor cell lines: murine melanoma (B16F10), human colon carcinoma (HT29), human breast adenocarcinoma (MCF-7), human cervical adenocarcinoma (HeLa), human hepatocellular liver carcinoma (HepG2) and human glioblastoma (MO59J, U343 and U251). The antiproliferative activity was evaluated using XTT assay and results were expressed as IC50. The most pronounced antiproliferative activity was observed for SM, with IC50 values ranging from 4.58 to 18.23 μg/mL. The lowest IC50 values were observed against HepG2, being 9.60 μg/mL for SL, 4.58 μg/mL for SM and 6.01 μg/mL for SS. Thus, SL, SM and SS demonstrated antiproliferative activity against the tumor cell lines tested, and were most effective against the HepG2 cell line.

The development of a "male pill" to control fertility is still a major challenge. Although women have several options to play an active role in the couple family planning, men are very limited in terms of contraceptive methods. Several approaches have been proposed to develop a male contraceptive and can be divided in two major groups: hormonal and non-hormonal methods. Within the testis, the somatic Sertoli cell (SC) is known as the "nurse cell" since it provides the physical and nutritional support for the developing germ cells. Moreover, adjacent SCs form the Sertoli/blood testis barrier (BTB), which divides the seminiferous epithelium into the basal and the apical compartments, controlling the passage of substances to the site where germ cells develop. Among the several functions of SC, its metabolism and the production of lactate, acetate and other metabolic factors are essential for the normal occurrence of spermatogenesis. In the last years, several works have highlighted that the metabolic cooperation established between SCs and developing germ cells is compromised in several diseases associated with male subfertility/infertility. Notably, several metabolismassociated proteins are specifically expressed in the testis. Thus, there are several evidences illustrating that the control of male fertility can be achieved by targeting testicular cells metabolism. Herein, we discuss the metabolic cooperation in testis as a potential pharmacological target to counteract subfertility/infertility promoted by several diseases, particularly metabolic diseases. We also discuss how it can contribute to the development of a male contraceptive.

Sertoli cells (SCs) glucose metabolism is crucial for spermatogenesis since developing germ cells consume lactate produced by SCs as their main energy source. Recently, androgens and estrogens have been implicated in SCs energy metabolism modulation, although the molecular mechanisms remained undisclosed. Here, we report the effect of sex steroid hormones on key points of cultured rat SCs glycolytic pathway. We used primary cultures of immature rat SCs treated with 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT). The transcript levels of glucose transporters (GLUTs), phosphofructokinase 1 (PFK-1) and lactate dehydrogenase C (LDH C) were analyzed after 25 and 50 h of culture by qPCR. Protein levels of GLUTs, PFK-1, LDH and monocarboxylate transporter 4 (MCT4) after 25 and 50 h were determined by western blot and LDH activity was also assessed. Our results show that both E2 and DHT downregulated the transcript levels of PFK-1, GLUT1 and GLUT3 after 50 h. However, only DHT-treated cells presented a downregulation of LDH C transcript levels. Interestingly, the protein levels of these enzymes and transporters remained unaltered except in DHT-treated cells that presented a significant decrease on GLUT1 protein levels evidencing a possible site for the regulation of SCs glucose metabolism by androgens. Taken together, our results provide evidence that sex steroid hormones action in SCs energy metabolism is mediated through modulation in glycolysis-related transporters and enzymes, particularly at the transcriptional level. DHT decreased GLUT1 protein levels and increased LDH activity after 25 h, evidencing key points for this hormone action in the regulation of SCs metabolism.

The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract. This includes the control of bicarbonate (HCO₃⁻) concentration, which plays an essential role in the maintenance of extracellular and intracellular pH (pH(i)) values. Diabetes mellitus alters pHi regulation in mammalian cells, mainly by altering the activity of ion transporters, particularly HCO₃⁻-dependent mechanisms. Yet, little is known about the effects of this pathology and its prodromal stage, prediabetes, on the membrane transport mechanisms of male reproductive tract cells. Herein, we analyzed protein and mRNA levels of the most relevant HCO₃⁻ transporters of the SLC4 family [anion exchanger 2 (AE2), Na⁺-driven Cl⁻/HCO₃⁻ exchanger (NDCBE), electrogenic Na⁺/HCO₃⁻ cotransporter 1 (NBCe1), electroneutral Na⁺/HCO₃⁻ cotransporter 1 (NBCn1)] in the testis and epididymis of a prediabetic animal model. Firstly, we identified the HCO₃⁻ transporters of the SLC4 family, in both testicular and epididymal tissue. Secondly, although no alterations were detected in protein expression, mRNA levels of NBCe1, NBCn1 and NDCBE were significantly increased in the testis of prediabetic rats. On the other hand, in the epididymis, prediabetes caused an increase of AE2 and a decrease of NDCBE protein levels. These alterations may be translated into changes of HCO₃⁻ transepithelial epididymal fluxes in vivo, which may represent a threat for sperm survival. Moreover, these results provide evidence of the molecular mechanism that may be responsible for the significant increase in abnormal sperm morphology already reported in prediabetic rats.

Freshwater bivalves are used as sentinel organisms to detect pollutants effects in the aquatic environment due to their sedentary nature, filter-feeding behaviour. We aimed to determine the in vivo, ex vivo and in vitro influence of Diethylstilbestrol (DES), a widely used synthetic non-steroidal estrogen and endocrine disruptor, in Anodonta cygnea shell growth mechanisms. For that, in vivo exposure to DES (0.75μM) during 15 days, in vitro and ex vivo exposure of outer mantle epithelium (OME) cells to DES (0.75μM), were performed followed by study of short-circuit current (Isc), transepithelial potential (Vt) and transepithelial conductance (Gt) as well as identification of membrane transport systems and intracellular pH (pHi). Our results show that in vivo exposure to DES decreases in 30% the OME Isc and ex vivo addition of DES to the basolateral side of OME also induced Isc decrease. Several membrane transporters such as V-type ATPases, Na(+)/H(+) exchangers, Na(+)-K(+) pump, Na(+)-driven and Na(+)-independent HCO3(-)/Cl(-) transporters and Na(+)/HCO3(-) co-transporter were identified as responsible for pHi maintenance in OME and noteworthy, DES caused a pHi decrease in OME cells similar to the effect observed when OME cells were exposed to 4,4'-diisothiocyanostilbene disulfonic acid (DIDS), an inhibitor of several bicarbonate membrane transporters. The addition of DIDS after OME cells exposure to DES did not cause any alteration. We concluded that DES is able to modulate membrane ion transport and pHi in the OME of A. cygnea and that this effect seems to be due to inhibition of HCO3(-)/Cl(-) co-transporters present on the basolateral membrane., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Insulin is essential for the regulation of glucose homeostasis. Insulin dysfunction occurs in several pathologies, such as diabetes mellitus, which is associated with fertility problems. Somatic Sertoli cells (SCs) not only metabolize glucose to lactate, which is the central energy source used by developing germ cells, but also determine the germ cell population size. If a deregulation in SCs apoptosis occurs, it will affect germ cells, compromising spermatogenesis. As SCs apoptotic signaling is a hormonally regulated process, we hypothesized that the lack of insulin could lead to alterations in apoptotic signaling. Therefore, we examined the effect of insulin deprivation on several markers of apoptotic signaling in cultured rat SCs. We determined mRNA and protein expression of apoptotic markers as well as caspase-3 activity. SCs cultured in insulin deprivation demonstrated a significant decrease on mRNA levels of p53, Bax, caspase-9, and caspase-3 followed by a significant increase of Bax and decrease of caspase-9 protein levels relatively to the control. Caspase-3 activity was also decreased in SCs cultured in insulin deprivation conditions. Our results show that insulin deprivation decreases caspase-dependent apoptotic signaling in cultured rat SCs evidencing a possible mechanism by which lack of insulin can affect spermatogenesis and fertility.

Sperm undergo maturation acquiring progressive motility and the ability to fertilize oocytes through exposure to the components of the epididymal fluid (EF). Although the establishment of a calcium (Ca(2+)) gradient along the epididymis has been described, its direct effects on epididymal function remain poorly explored. Regucalcin (RGN) is a Ca(2+)-binding protein, regulating the activity of Ca(2+)-channels and Ca(2+)-ATPase, for which a role in male reproductive function has been suggested. This study aimed at comparing the morphology, assessed by histological analysis, and function of epididymis, by analysis of sperm parameters, antioxidant potential and Ca(2+) fluxes, between transgenic rats overexpressing RGN (Tg-RGN) and their wild-type littermates. Tg-RGN animals displayed an altered morphology of epididymis and lower sperm counts and motility. Tissue incubation with (45)Ca(2+) showed also that epididymis of Tg-RGN displayed a diminished rate of Ca(2+)-influx, indicating unbalanced Ca(2+) concentrations in the epididymal lumen. Sperm viability and the frequency of normal sperm, determined by the one-step eosin-nigrosin staining technique and the Diff-Quik staining method, respectively, were higher in Tg-RGN. Moreover, sperm of Tg-RGN rats showed a diminished incidence of tail defects. Western blot analysis demonstrated the presence of RGN in EF as well as its higher expression in the corpus region. The results presented herein demonstrated the importance of maintaining Ca(2+)-levels in the epididymal lumen and suggest a role for RGN in sperm maturation. Overall, a new insight into the molecular mechanisms driving epididymal sperm maturation was obtained, which could be relevant to development of better approaches in male infertility treatment and contraception.

The purpose of this study was to determine the effects of 2,4-D, an herbicide used worldwide also known as endocrine disruptor, in Sertoli cell (SC) metabolism. Immature rat SCs were maintained 50h under basal conditions or exposed to 2,4-D (100nM, 10μM and 1mM). SCs exposed to 10μM and 1mM of 2,4-D presented lower intracellular glucose and lactate content. Exposure to 10μM of 2,4-D induced a significant decrease in glucose transporter-3 mRNA levels and phosphofructokinase-1 mRNA levels decreased in cells exposed to 100nM and 10μM of 2,4-D. Exposure to 100nM and 10μM also induced a decrease in lactate dehydrogenase (LDH) mRNA levels while the LDH protein levels were only decreased in cells exposed to 1mM of 2,4-D. Exposure to 2,4-D altered glucose uptake and metabolization in SCs, as well as lactate metabolism and export that may result in impaired spermatogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Usnic acid is one of the most common and abundant metabolites found in various lichen genera, which are important sources of biologically active compounds. The aim of this study was to evaluate the genotoxic and antigenotoxic potential of (+)-usnic acid (UA) by the micronucleus and comet assays in V79 cell cultures and Swiss mice. For assessment of genotoxicity, V79 cells were treated with 15, 30, 60, and 120μg/mL UA, established based on clonogenic efficiency cytotoxic assay. Swiss mice were treated with UA doses of 25, 50, 100, and 200mg/kg body weight. The same concentrations of UA were combined with methyl methanesulfonate (MMS) for evaluation of antigenotoxicity. The in vitro results demonstrated that UA induced DNA damage at concentrations of 60 and 120μg/mL in the comet assay. However, no genotoxic effect was observed in the micronucleus test using V79 cells at the concentrations tested. No genotoxic effects were observed for the different UA treatments in in vivo test system. Combined administration of UA and MMS significantly reduced the frequencies of micronuclei and DNA damage in vitro and in vivo when compared to treatment with MMS alone. Although the mechanisms underlying the protective effect of UA are not completely understood, the antioxidant activity of this metabolite may explain its protective effect against MMS-induced genotoxicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Diabetes mellitus (DM) is one of the greatest public health threats in modern societies. Although during a few years it was suggested that DM had no significant effect in male reproductive function, this view has been challenged in recent years. The increasing incidence of DM worldwide will inevitably result in a higher prevalence of this pathology in men of reproductive age and subfertility or infertility associated with DM is expected to dramatically rise in upcoming years. From a clinical perspective, the evaluation of semen parameters, as well as spermatozoa deoxyribonucleic acid (DNA) integrity, are often studied due to their direct implications in natural and assisted conception. Nevertheless, recent studies based on the molecular mechanisms beyond glucose transport in testicular cells provide new insights in DM-induced alterations in male reproductive health. Testicular cells have their own glucose sensing machinery that react to hormonal fluctuations and have several mechanisms to counteract hyper- and hypoglycemic events. Moreover, the metabolic cooperation between testicular cells is crucial for normal spermatogenesis. Sertoli cells (SCs), which are the main components of blood-testis barrier, are not only responsible for the physical support of germ cells but also for lactate production that is then metabolized by the developing germ cells. Any alteration in this tied metabolic cooperation may have a dramatic consequence in male fertility potential. Therefore, we present an overview of the clinical significance of DM in the male reproductive health with emphasis on the molecular mechanisms beyond glucose fluctuation and transport in testicular cells., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Diabetes mellitus is a metabolic disorder that may arise from diet habits and is growing to epidemic proportions. Young male diabetic patients present high infertility/subfertility prevalence resulting from impaired reproductive function and poor semen quality. We aimed to evaluate the effects of a high-energy diet (HED) on glucose tolerance/insulin levels and correlate the observed effects on male reproductive function with overall testicular metabolism. After 1 month, HED fed rats showed increased glycaemic levels, impaired glucose tolerance and hypoinsulinaemia. Moreover, an imbalance of intratesticular and serum testosterone levels was observed, whereas those of 17β-estradiol were not altered. High-energy diet also affected the reproductive parameters, with HED rats exhibiting a significant increase in abnormal sperm morphology. Glycolytic metabolism was favoured in testicles of HED rats with an increased expression of both glucose transporters 1 (GLUT1) and 3 (GLUT3) and the enzyme phosphofrutokinase 1. Moreover, lactate production and the expression of metabolism-associated genes and proteins involved in lactate production and transport were also enhanced by HED. Alanine testicular content was decreased and thus intratesticular lactate/alanine ratio in HED rats was increased, suggesting increased oxidative stress. Other energetic substrates such as acetate and creatine were not altered in testis from HED rats, but intratesticular glycine content was increased in those animals. Taken together, these results suggest that HED induces a pre-diabetic state that may impair reproductive function by modulating overall testicular metabolism. This is the first report on testicular metabolic features and mechanisms related with the onset of a pre-diabetic state., (© 2013 American Society of Andrology and European Academy of Andrology.)

Apoptosis is an important regulatory event in testicular homeostasis and optimization of sperm production. Sertoli cells (SCs) form the blood-testis barrier creating a special microenvironment where germ cells develop and are under strict hormonal control. Estrogens and androgens are known to play critical roles in SCs functioning, improving their in vitro survival by preventing apoptotic progression. Herein, we studied the influence of 17β-estradiol (E2) and 5α-dihydrotestosterone (DHT) on the apoptotic signaling pathways of immature rat cultured SCs. For that we chose key points of the apoptotic pathway that interact with the mitochondria and evaluated the mRNA expression and/or protein levels of several apoptotic markers such as p53, the anti-apoptotic protein Bcl2, the pro-apoptotic Bcl2 family member Bax, the apoptosis-inducing factor (AIF) and caspase-3 and 9. Caspase-3 activity and DNA fragmentation were also evaluated as endpoint markers of apoptosis. E2 and DHT down-regulated the mRNA transcript levels of p53, Bax, caspase-9 and caspase-3. The protein levels of AIF were reduced after DHT treatment while E2-treated cells presented decreased levels of cleaved caspase-9 protein. Moreover, Bax/Bcl2 ratio was significantly decreased in E2-treated cells. The apoptotic endpoints caspase-3 activity and DNA fragmentation presented significant decreased levels after hormonal treatment. Taken together, these results show that E2 and DHT act as apoptotic signaling modulators in in vitro immature rat SCs suggesting that androgens and estrogens may be capable of modulating independent pathways of the apoptotic event by regulating different pro-apoptotic factors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

(-)-Cubebin (CUB) is a lignan isolated from dry seeds of Piper cubeba. We aimed to assess its genotoxic potential and influence on chromosomal damage (frequency of micronuclei - MN) induced by doxorubicin (DXR) in V79 cells and by urethane (URE) in somatic Drosophila melanogaster cells. Our findings indicate an absence of a CUB-mediated genotoxic effect at the concentrations tested. The results also revealed that CUB significantly reduced the frequency of MN induced by DXR, with a mean reduction of 63.88%. In a previous study, our research group demonstrated an absence of CUB-mediated mutagenic effects through the wing Somatic Mutation and Recombination Test (SMART) in Drosophila. In the present study, we used the standard and high bioactivation versions of the SMART to estimate the antigenotoxic effects of CUB associated with URE. At lower concentrations, the recombination level decreased, but at the highest concentration, the recombination level increased. Our data and previous studies suggest that CUB may act as a free radical scavenger at low concentrations, a pro-oxidant at higher concentrations when it interacts with the enzymatic system that catalyzes the metabolic detoxification of DXR or URE, and/or an inducer of recombinational DNA repair., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Blood testis barrier (BTB) is one of the tightest blood-barriers controlling the entry of substances into the intratubular fluid. Diabetes Mellitus (DM) is an epidemic metabolic disease concurrent with falling fertility rates, which provokes severe detrimental BTB alterations. It induces testicular alterations, disrupting the metabolic cooperation between the cellular constituents of BTB, with dramatic consequences on sperm quality and fertility. As Sertoli cells are involved in the regulation of spermatogenesis, providing nutritional support for germ cells, any metabolic alteration in these cells derived from DM may be responsible for spermatogenesis disruption, playing a crucial role in fertility/subfertility associated with this pathology. These cells have a glucose sensing machinery that reacts to hormonal fluctuations and several mechanisms to counteract hyper/hypoglycemic events. The role of DM on Sertoli/BTB glucose metabolism dynamics and the metabolic molecular mechanisms through which DM and insulin deregulation alter its functioning, affecting male reproductive potential will be discussed.

Hormonal regulation is essential to spermatogenesis. Sertoli cells (SCs) have functions that reach far beyond the physical support of germ cells, as they are responsible for creating the adequate ionic and metabolic environment for germ cell development. Thus, much attention has been given to the metabolic functioning of SCs. During spermatogenesis, germ cells are provided with suitable metabolic substrates, in a set of events mediated by SCs. Multiple signaling cascades regulate SC function and several of these signaling pathways are hormone-dependent and cell-specific. Within the seminiferous tubules, only SCs possess receptors for some hormones rendering them major targets for the hormonal signaling that regulates spermatogenesis. Although the mechanisms by which SCs fulfill their own and germ cells metabolic needs are mostly studied in vitro, SC metabolism is unquestionably a regulation point for germ cell development and the hormonal control of these processes is required for a normal spermatogenesis.

Bicarbonate (HCO₃⁻) membrane transport systems are crucial players in the physiology of several tissues. The molecular basis of HCO₃⁻ membrane transport is of major physiological relevance since this ion is involved in the establishment of intracellular and extracellular ionic composition, osmolariy and pH. The membrane HCO₃⁻ transporters are divided in two main families: solute carrier 4 (SLC4) and solute carrier 26 (SLC26), although HCO₃⁻ concentration can also be regulated by the cystic fibrosis transmembrane regulator (CFTR). In most tissues the SLC4 family represents the majority of HCO₃⁻ transporters members, which can be divided in two subgroups: the Na⁺-dependent and the Na⁺-independent transporters. The SLC26 family consists of ten members that can transport diverse ions besides HCO₃⁻. In the male reproductive tract, HCO₃⁻ transport occurs in several processes in order to assure a correct pursuance of the spermatogenetic event and spermatozoa capacitation, being also necessary for egg fertilization. Indeed, the formation of competent spermatozoa, the maintenance of an adequate ductal luminal milieu and spermatozoa capacitation are highly dependent of ionic balance and pH. Perturbations in these processes result in reduced male reproductive health and consequently male subfertility and/or infertility. Thus, it is imperative to understand HCO₃⁻ transport dynamics in order to identify and counteract possible alterations related with reduced male fertility caused by pathological conditions. Herein, we will review the major families and subfamilies of HCO₃⁻ membrane transport, discussing the molecular basis of HCO₃⁻ transport in the male reproductive tract and its role in male-associated subfertility and/or infertility.

Artepillin C (3,5-diprenyl-p-coumaric acid) is one of the major phenolic compounds found in Brazilian green propolis, as well as in its botanical source, Baccharis dracunculifolia DC (Asteraceae). The present study evaluated the possible genotoxic and protective activities of artepillin C, in vitro, using methyl methanesulfonate (MMS) as a positive control, by comet and micronucleus assays. The cultures of Chinese hamster lung fibroblasts (V79 cells) were treated with different concentrations of artepillin C (2.5, 5.0, 10.0, and 20 μM). In antigenotoxicity assessment, the 3 concentrations of artepillin C (2.5, 5.0, and 10.0 μM) were associated with MMS (200 μM-comet assay and 400 μM-micronucleus assay). A statistically significant increase in the DNA damage and micronucleus frequencies was observed in the culture treated with the highest concentration of the artepillin C in comparison to the control group. All concentrations of artepillin C showed protective activity in relation to MMS-induced genotoxicity, which may be due to its antioxidant properties.

The development of leukemia and lymphomas is related to the increase in inflammatory process modulators. These, in turn, have divergent actions on the neoplastic process. Populations of T cells have different roles in the neoplastic environment; while interferon-gamma positive T cells have antitumor activity, the FoxP3+interleukin-10 positive population present a pro-tumor activity. Simultaneously, the inflammatory process promotes the mobilization of fatty acids from the cell membrane to produce lipid mediators, which also participate of the inflammatory response. Eicosapentaenoic (EPA) and docosahexaenoic (DHA) omega-3 fatty acids, when incorporated in the plasmatic membrane, decrease the arachidonic acid (AA) metabolism and the production of eicosanoids derived from it. Thus, an alternative family of lipid mediators are produced that are often less inflammatory than those produced from arachidonic acid. Fatty acids can also influence the production of peptide mediators such as cytokines, and the expression of transcription factors, which can determine the production patterns of eicosanoids and cytokines as well as cell differentiation. Due to these properties, the objective of this literature review was to investigate studies published over the last 15 years on the effects of using omega-3 fatty acids on inflammatory markers in leukemia and lymphomas.

Since the beginning of propolis research, several groups have studied its antibacterial, antifungal, and antiviral properties. However, most of these studies have only employed propolis ethanolic extract (PEE) leading to little knowledge about the biological activities of propolis water extract (PWE). Based on this, in a previous study, we demonstrated the anti-inflammatory and immunomodulatory activities of PWE. In order to better understand the equilibrium between effectiveness and toxicity, which is essential for a new medicine, the characteristics of PWE were analyzed. We developed and validated an RP-HPLC method to chemically characterize PWE and PEE and evaluated the in vitro antioxidant/antimicrobial activity for both extracts and the safety of PWE via determining genotoxic potential using in vitro and in vivo mammalian micronucleus assays. We have concluded that the proposed analytical methodology was reliable, and both extracts showed similar chemical composition. The extracts presented antioxidant and antimicrobial effects, while PWE demonstrated higher antioxidant activity and more efficacious for the most of the microorganisms tested than PEE. Finally, PWE was shown to be safe using micronucleus assays.

Cellular systems implanted into an injured nerve may produce growth factors or extracellular matrix molecules, modulate the inflammatory process and eventually improve nerve regeneration. In the present study, we evaluated the therapeutic value of human umbilical cord matrix MSCs (HMSCs) on rat sciatic nerve after axonotmesis injury associated to Vivosorb® membrane. During HMSCs expansion and differentiation in neuroglial-like cells, the culture medium was collected at 48, 72 and 96 h for nuclear magnetic resonance (NMR) analysis in order to evaluate the metabolic profile. To correlate the HMSCs ability to differentiate and survival capacity in the presence of the Vivosorb® membrane, the [Ca(2+)]i of undifferentiated HMSCs or neuroglial-differentiated HMSCs was determined by the epifluorescence technique using the Fura-2AM probe. The Vivosorb® membrane proved to be adequate and used as scaffold associated with undifferentiated HMSCs or neuroglial-differentiated HMSCs. In vivo testing was carried out in adult rats where a sciatic nerve axonotmesis injury was treated with undifferentiated HMSCs or neuroglial differentiated HMSCs with or without the Vivosorb® membrane. Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index (SFI), extensor postural thrust (EPT), and withdrawal reflex latency (WRL). Stereological analysis was carried out on regenerated nerve fibers. In vitro investigation showed the formation of typical neuroglial cells after differentiation, which were positively stained for the typical specific neuroglial markers such as the GFAP, the GAP-43 and NeuN. NMR showed clear evidence that HMSCs expansion is glycolysis-dependent but their differentiation requires the switch of the metabolic profile to oxidative metabolism. In vivo studies showed enhanced recovery of motor and sensory function in animals treated with transplanted undifferentiated and differentiated HMSCs that was accompanied by an increase in myelin sheath. Taken together, HMSC from the umbilical cord Wharton jelly might be useful for improving the clinical outcome after peripheral nerve lesion., (Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Blood-tissue barriers prevent an uncontrolled exchange of large molecules between adjacent but metabolically separated compartments. There are several known barriers and two of the most important and tightest blood-tissue barriers are the blood-testis barrier (BTB) and the blood-brain barrier (BBB). Under normal conditions these barriers, formed by tight junctions between adjacent cells, control the entry of substances and metabolites. However, hyperglycemia and other diabetes-related complications, such as hypertension, impair the function of these biological barriers with dramatic consequences. Although both, BBB and BTB, are responsible for the maintenance of different biological processes, they have some remarkable similarities not always explored when looking at metabolic-related diseases such as diabetes. These barriers possess their own glucose sensing machinery, suffer a tied hormonal control and have specific mechanisms to counteract hyper- and hypoglycemia. In BBB and BTB the insulin signaling is also distinct from other tissues and organs thus evidencing their importance in protecting against or exacerbating the effects of diabetes on glucose metabolism. The control of glucose and lactate levels in brain and testis highlights the role of these barriers in protecting against peripheral glucose and lactate fluctuations that occur in the diabetic individual. We review the role of BBB and BTB in the control of glucose and metabolic dysfunction caused by diabetes in the brain and seminiferous epithelium. Gaining a better understanding of the molecular mechanisms through which glucose metabolism disrupts BBB and BTB function may highlight new opportunities for the treatment of diabetic complications in brain and male reproductive function.

Prostate cancer (PCa) progresses from an early stage, confined to prostate, to a more aggressive metastasized cancer related with loss of androgen responsiveness. Although, it has been recognized that PCa cells have unique metabolic features, their glycolytic profile in androgen-dependent and androgen-independent stages of disease is much less known. Hence, the main purpose of this study was to compare glucose metabolism in androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) PCa cells. Cell culture medium was collected and differences in glucose consumption and, lactate and alanine production were measured using Proton Nuclear Magnetic Resonance ((1)H NMR) spectra analysis. The mRNA and protein expression of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK1), lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) were determined by real-time PCR and Western Blot, respectively. The obtained results demonstrate that androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) cells consumed similar amounts of glucose, whereas PC3 cells present higher lactate production. This increase in lactate production was concomitant with higher levels of MCT4 protein, increased LDH activity and higher lactate/alanine ratio, also suggesting increased levels of oxidative stress in PC3 cells. However, protein levels of LDH, associated with lactate metabolism, and GLUT3, involved in glucose uptake, were decreased in PC3 comparatively with LNCaP. Androgen-responsive and nonresponsive PCa cells present distinct glycolytic metabolism profiles, which suggest that targeting LDH and MCT4 metabolic pathways may be an important step for the development of new diagnostic and therapeutic strategies in the different stages of PCa., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Solanum lycocarpum St.-Hil (Solanaceae) is a hairy shrub or small much-branched tree of the Brazilian Cerrado, popularly known as "fruit-of-wolf". Considering that the induction of chromosomal mutations is involved in the process of carcinogenesis, and that S. lycocarpum is often used in folk medicine, it becomes relevant to study its effect on genetic material. In this sense, the aim of present study was to determine the possible cytotoxic, genotoxic and antigenotoxic potentials of S. lycocarpum fruits glycoalkaloid extract (SL) in Chinese hamster lung fibroblasts (V79 cells). The cytotoxicity was evaluated by the colony forming assay, apoptosis and necrosis assay, Trypan blue exclusion dye method and mitotic index. Genotoxic and antigenotoxic potential were evaluated by comet and chromosomal aberrations assays. Four concentrations of SL (4, 8, 16 and 32 μg/mL) were used for the evaluation of its genotoxic potential. The DNA damage-inducing agent methyl methanesulfonate (MMS, 22 μg/mL) was utilized in combination with extract to evaluate a possible protective effect. The results showed that SL was cytotoxic at concentrations above 32 μg/mL by the colony forming assay. For apoptosis and necrosis assay, the concentration of 64 μg/mL of SL showed statistically significant increase in cell death by apoptosis and necrosis, while the concentrations of 128 and 256 μg/mL of SL demonstrated statistically significant increase in cell death by necrosis, compared with the control group. Analysis of cell viability by Trypan blue exclusion indicated >96% viability for treatments with concentrations up to 32 μg/mL of SL. No significant differences in MI were observed between cultures treated with different concentrations of SL (4, 8, 16 and 32 μg/mL) alone or in combination with MMS and the negative control, indicating that these treatments were not cytotoxic. The comet and chromosomal aberrations assays revealed that SL does not display genotoxic activity. Moreover, the different concentrations of SL showed protective effect against both genomic and chromosomal damages induced by MMS., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Spiders are exceptionally diverse and abundant organisms in terrestrial ecosystems and their evolutionary success is certainly related to their capacity to produce different types of silks during their life cycle, making a specialized use on each of them. Presenting particularly tandemly arranged amino acid repeats, silk proteins (spidroins) have mechanical properties superior to most synthetic or natural high-performance fibers, which makes them very promising for biotechnology industry, with putative applications in the production of new biomaterials. During the evolution of spider species, complex behaviors of web production and usage have been coupled with anatomical specialization of spinning glands. Spiders retaining ancestral characters, such as the ones belonging to the Mygalomorph group, present simpler sorts of webs used mainly to build burrows and egg sacs, and their silks are produced by globular undifferentiated spinning glands. In contrast, Araneomorphae spiders have a complex spinning apparatus, presenting up to seven morphologically distinct glands, capable to produce a more complex set of silk polymers with different degrees of rigidness and elasticity associated with distinct behaviors. Aiming to provide a discussion involving a number of spider silks' biological aspects, in this review we present descriptions of members from each family of spidroin identified from five spider species of the Brazilian biodiversity, and an evolutionary study of them in correlation with the anatomical specialization of glands and spider's spinning behaviors. Due to the biotechnological importance of spider silks for the production of new biomaterials, we also discuss about the new possible technical and biomedical applications of spider silks and the current status of it.

Background: Several important functions for a successful spermatogenesis are dependent on Sertoli cells (SCs). Besides their unique characteristics as support cells, they produce essential cofactors and metabolites, and are responsible for nurturing the developing germ cells. The continuous production of lipids, phospholipids and proteins by germ cells must require high amounts of metabolic precursors. Thus, we hypothesized that hSCs could produce acetate in a hormonally-regulated manner., Methods: hSC-enriched primary cultures were maintained in the absence of insulin or in the presence of 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT). Acetate production was determined by 1H-NMR. mRNA gene expression levels of Acetyl CoA hydrolase (ACoA Hyd) and Acetyl CoA synthase (ACoA Synt) were determined by RT-PCR., Results: hSCs produced high amounts of acetate suggesting that this metabolite should play a key role on the progression of spermatogenesis, namely as a metabolic precursor for the synthesis of cellular constituents. In addition, acetate metabolism proved to be under strict hormonal regulation. In the presence of E2 or DHT, hSCs produced different amounts of acetate. While E2 treatment increased acetate production, increasing ACoA Hyd gene transcript levels, DHT-treated cells showed decreased acetate production, differently modulating the ratio ACoA Hyd/ACoA Synt. Surprisingly, insulin-deprivation completely suppressed acetate production/export and significantly decreased the ACoA Hyd gene transcript levels., General Significance: Taken together, these results suggest that, although hSCs are primarily described as lactate producers, the elevated production of acetate deserves special attention, in order to clarify the mechanisms behind its hormonal regulation and its role on a successful spermatogenesis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Baccharin is one of the major chemical compounds isolated from the aerial parts of Baccharis dracunculifolia DC (Asteraceae), a native plant of South America and the most important botanical source of the Brazilian green propolis that has been used in alternative medicine to treat inflammation, liver disorders, and stomach ulcers. The present study was carried out in V79 cells to determine the possible genotoxic and antigenotoxic activities of baccharin utilizing comet and micronucleus assays, where 2 known mutagenic agents with different mechanisms of DNA damage were used as positive controls. The V79 cells were treated with concentrations of baccharin (0.25, 0.5, 1.0, and 2.0 μg/mL) and for to investigate the antigenotoxicity these concentrations were associated with methyl methanesulfonate (MMS; 200 μM-comet assay and 400 μM-micronucleus assay) or hydrogen peroxide (H(2)O(2;) 50 μM-comet assay and 100 μM-micronucleus assay). Statistically significant differences in the rate of DNA damage were observed in cultures treated with the highest concentration of baccharin when compared to the control group, but this difference was not found in the micronucleus assay. The results also showed that the frequencies of DNA damage and micronuclei induced by MMS and H(2)O(2) were significantly reduced after treatment with baccharin. The baccharin showed a chemoprevention effect and can be the chemical compound responsible for the antigenotoxicity also demonstrated by the B. dracunculifolia. The antioxidant potential of baccharin may be related to its chemoprevention activity induced against both genomic and chromosomal damages., (© 2012 Institute of Food Technologists®)