Alberto Barros

University of Porto, Oporto, Porto, Portugal

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Publications (197)530.82 Total impact

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    ABSTRACT: A pioglitazona é um potente agonista sintético dos recetores ativados por proliferadores de peroxissoma gama utilizado para tratar a Diabetes Mellitus tipo 2 (DMT2). Outros benefícios para a utilização da pioglitazona têm sido reportados mas os seus efeitos na reprodução masculina permanecem desconhecidos. Além da pioglitazona, a metformina é uma biguanida usada como primeira linha no tratamento da DMT2 (1). Ambos são utilizados na prática clínica como terapia combinada. A cooperação metabólica entre as células testiculares é crucial para a ocorrência de uma espermatogénese normal (2). A célula de Sertoli (CS) absorve glicose e converte-a em lactato, que é exportado e utilizado como fonte energética pelas células germinativas em desenvolvimento. O objetivo deste trabalho foi investigar os efeitos da pioglitazona, isoladamente ou em combinação com a metformina, no metabolismo das CSs. As CSs humanas foram cultivadas na ausência e na presença de pioglitazona (1, 10, 100 µM e 1,5 µM + 10 µM metformina). Os níveis de proteína de fosfofrutoquinase 1 (PFK1), lactato desidrogenase (LDH), transportador de monocarboxilato 4 (MCT4) e transportadores de glicose (GLUT1, GLUT2 e GLUT3) foram determinados por Western blot e Slot blot. A atividade de LDH foi determinada e a produção e consumo de metabolitos foram determinados por 1 H-RMN. Os resultados demonstram que a concentração farmacológica de pioglitazona não altera o suporte nutricional da espermatogénese, mas a concentração suprafarmacológica pode resultar na diminuição da produção de lactato necessário para as células germinativas, o que pode comprometer o potencial reprodutor no homem.
    Full-text · Conference Paper · Jan 2016
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    ABSTRACT: Ação da leptina no metabolismo da glucose das células de Sertoli: impacto na fertilidade masculina Nos últimos anos os hábitos alimentares e o estilo de vida têm vindo a tornar-se pouco saudáveis, favorecendo o desenvolvimento de doenças metabólicas (por exemplo Diabetes tipo 2 e obesidade) (1). A leptina é uma hormona produzida no tecido adiposo responsável pela sensação de saciedade. Os níveis desta hormona estão positivamente relacionados com a quantidade de gordura corporal e têm sido descritos efeitos negativos associados ao aumento da sua concentração na função reprodutora masculina (2). No entanto, os mecanismos moleculares pelos quais a leptina afeta a fertilidade masculina permanecem desconhecidos. Neste estudo testámos a hipótese da leptina alterar o metabolismo das células de Sertoli, que são responsáveis pelo suporte nutricional da espermatogénese (3). Células de Sertoli humanas (hSCs) foram expostas a doses crescentes de leptina (5, 25, 50 ng/ml) e posteriormente foram determinados os níveis de proteína dos transportadores de glucose (GLUT1, GLUT2 e GLUT3), da fosfofructocinase, da lactato desidrogenase (LDH) e do transportador de ácidos monocarboxílicos 4 (MCT4). Foi também determinada a atividade da LDH e o consumo/produção de metabolitos por ressonância magnética nuclear de protão. Identificámos o recetor da leptina, pela primeira vez, em hSCs. Demonstrámos que a expressão proteica de GLUT2 e a atividade da LDH são moduladas pela leptina assim como a produção de acetato pelas hSCs. Este é o primeiro trabalho que demonstra que a leptina afeta o suporte nutricional da espermatogénese através das hDCs. Esta ação direta da leptina nas HSCs representa um novo mecanismo pelo qual a obesidade pode induzir infertilidade nos homens.
    Full-text · Conference Paper · Jan 2016
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    ABSTRACT: INTRODUÇÃO E OBJETIVOS A infertilidade afeta milhões de casais em todo mundo, sendo que um terço desses casos resulta de problemas associados ao sexo masculino (1). Um grande número de casos de infertilidade masculina tem origem idiopática, em que os pacientes apresentam anormalidades inexplicáveis com alteração nos parâmetros reprodutivos. A desregulação dos níveis plasmáticos de estrogénios (E2) tem sido associada a casos de infertilidade masculina. Vários estudos apresentam evidências de que o E2 desempenha um papel essencial ao nível da espermatogénese e da função reprodutiva masculina (2). O E2 está também implicado na regulação da expressão de transportadores iónicos no trato reprodutor masculino (3), em que o controlo adequado do conteúdo iónico no meio luminal desempenha um papel crucial. O bicarbonato (HCO 3-) é essencial não apenas para a homeostase iónica mas também para manter o pH ao longo do trato reprodutor masculino (4). Tem sido demonstrado a expressão de alguns transportadores de HCO3- no trato reprodutor masculino e a sua alteração em casos patológicos (5). O presente trabalho teve como objetivo identificar três dos mais relevantes transportadores membranares de HCO3- dependentes de Na+ em células de Sertoli humanas (hSCs) e avaliar o efeito do E2 na expressão e funcionalidade desses transportadores. MÉTODOS Foram usadas biopsias testiculares obtidas a partir de pacientes com espermatogénese conservada, após o seu consentimento informado por escrito. A partir das biopsias testiculares, isolaram-se as hSCs como descrito por Oliveira e colaboradores (2009) (6), que foram incubadas a 33ºC numa atmosfera com 6% CO2: 94% O2. Após atingirem uma confluência de cerca de 90%-95%, as hSCs foram separadas em grupo controlo e grupo tratado com E2 (100 nM). Estas culturas de hSCs foram usadas para estudar a localização subcelular (lado apical ou basal) dos três transportadores de HCO3- (NDCBE, NBCe1 e NBCn1) por microscopia confocal. Avaliaram-se os efeitos do E2 na expressão e funcionalidade dos transportadores por imunodetecção, epifluorescência e estudos de voltagem controlada, após a exposição a E2 (100 nM) durante 24 horas. A análise estatística dos resultados foi feita por T-test ou ANOVA. RESULTADOS Identificou-se pela primeira vez os três transportadores estudados (NBCn1, NBCe1 e NDCBE) em hSCs. O NBCn1 e o NBCe1 foram localizados na porção basal da membrana destas células, enquanto o NDCBE foi detetado na região apical da membrana das hSC polarizadas. Observou-se que a exposição a E2 provocou um aumento nos níveis proteicos de NBCn1, NBCe1 e NDCBE, assim como alterações no pH intracelular e no transporte transcelular. O E2 também provocou perturbações na corrente de curto-circuito induzida por ATP e alterações no pH intracelular. CONCLUSÕES Em suma, demonstrou-se a relação entre o aumento dos níveis de E2 e da expressão/função do NBCn1, NBCe1 e NDCBE nas hSC, fornecendo novas evidências sobre os mecanismos pelos quais o E2 pode regular a fisiologia destas células e consequentemente a espermatogénese, com influência direta sobre o potencial reprodutivo masculino.
    Full-text · Conference Paper · Jan 2016
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    ABSTRACT: A cafeína é um componente comum do café, chá, bebidas energéticas, produtos que contenham cacau ou chocolate e até mesmo de vários medicamentos. O consumo excessivo de cafeína tem sido associado a diversos problemas de foro reprodutivo. Além disso, foi demonstrado que este composto tem uma ação moduladora no metabolismo celular. Isto levou-nos a colocar a hipótese de a cafeína poder alterar o metabolismo das células de Sertoli humanas (hSCs), as quais são essenciais para a espermatogénese e para a fertilidade masculina. Para verificar esta hipótese, as hSCs foram cultivadas na presença de cafeína (5, 50 e 500 µM) e o seu perfil glicolítico foi avaliado através do estudo do consumo de glucose e da produção de lactato e alanina. Os níveis de expressão proteica dos transportadores de glucose (GLUT1 e GLUT3), fosfofrutoquinase 1 (PFK1), lactato desidrogenase (LDH) e transportador de monocarboxilatos 4 (MCT4) foram também determinados, assim como a atividade da enzima LDH. Para além disso, como a cafeína demonstrou possuir propriedades antioxidantes, avaliámos a capacidade antioxidante das hSCs após a exposição à cafeína, assim como a formação de grupos carbonilo e a peroxidação lipídica. A cafeína nas concentrações mais baixas (5 e 50 µM) estimulou a produção de lactato, mas apenas as hSCs expostas a uma concentração de 50 µM apresentaram um aumento da expressão dos GLUTs. Na concentração mais elevada (500 µM), a atividade da LDH foi estimulada de forma a sustentar a produção de lactato. Notavelmente, a capacidade antioxidante das hSCs diminuiu de uma forma dose-dependente. Os nossos resultados indicam claramente que a exposição das hSCs à cafeína altera o seu metabolismo celular. Além da estimulação da produção de lactato nas hSCs expostas à cafeína, o qual é essencial para o desenvolvimento e sobrevivência das células germinativas, o perfil oxidativo destas células foi alterado. Adicionalmente, a concentração mais elevada de cafeína (500 μM) induziu um estado pró-oxidante, o que pode ser deletério para a progressão da espermatogénese, uma vez que esta normalmente já envolve níveis elevados de stress oxidativo. Mais estudos serão necessários para clarificar que doses de cafeína serão benéficas/prejudiciais para a função das hSCs, a fim de evitar problemas a nível da reprodução masculina.
    Full-text · Conference Paper · Jan 2016
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    ABSTRACT: Objective: To study the role of mammalian target of rapamycin (mTOR) in the regulation of human Sertoli cell (hSC) metabolism, mitochondrial activity, and oxidative stress. Design: Experimental study. Setting: University research center and private IVF centers. Patient(s): Six men with anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis. Intervention(s): Testicular biopsies were used from patients under treatment for recovery of male gametes. Primary hSCs cultures were established from each biopsy and divided into a control group and one treated with rapamycin, the inhibitor of the mammalian target of mTOR, for 24 hours. Main outcome measure(s): Cytotoxicity of hSCs to rapamycin was evaluated by sulforhodamine B assay. The glycolytic profile of hSCs was assessed by proton nuclear magnetic resonance and by studying protein expression of key glycolysis-related transporters and enzymes. Expression of mitochondrial complexes and citrate synthase activity were determined. Protein carbonylation, nitration, lipid peroxidation, and sulfhydryl protein group contents were quantified. The mTOR signaling pathway was studied. Result(s): Rapamycin increased glucose consumption by hSCs, maintaining lactate production. Alanine production by rapamycin-exposed hSCs was affected, resulting in an unbalanced intracellular redox state. Rapamycin-exposed hSCs had decreased expression of mitochondrial complex III and increased lipid peroxidation, whereas other oxidative stress markers were unaltered. Treatment of hSCs with rapamycin down-regulated phospho-mTOR (Ser-2448) levels, illustrating an effective partial inhibition of mTORC1. Protein levels of downstream signaling molecule p-4E-BP1 were not altered, suggesting that during treatment it became rephosphorylated. Conclusion(s): We show that mTOR regulates the nutritional support of spermatogenesis by hSCs and redox balance in these cells.
    Full-text · Article · Dec 2015 · Fertility and sterility
  • Rute Pereira · Rosália Sá · Alberto Barros · Mário Sousa
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    ABSTRACT: The genetic bases and molecular mechanisms involved in the assembly and function of the flagellum components as well as in the regulation of the flagellar movement are not fully understood, especially in humans. There are several causes for sperm immotility, of which some can be avoided and corrected, whereas other are related to genetic defects and deserve full investigation to give a diagnosis to patients. This review was performed after an extensive literature search on the online databases PubMed, ScienceDirect, and Web of Science. Here, we review the involvement of regulatory pathways responsible for sperm motility, indicating possible causes for sperm immotility. These included the calcium pathway, the cAMP-dependent protein kinase pathway, the importance of kinases and phosphatases, the function of reactive oxygen species, and how the regulation of cell volume and osmolarity are also fundamental components. We then discuss main gene defects associated with specific morphological abnormalities. Finally, we slightly discuss some preventive and treatments approaches to avoid development of conditions that are associated with unspecified sperm immotility. We believe that in the near future, with the development of more powerful techniques, the genetic causes of sperm immotility and the regulatory mechanisms of sperm motility will be better understand, thus enabling to perform a full diagnosis and uncover new therapies.
    No preview · Article · Dec 2015 · Asian Journal of Andrology
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    ABSTRACT: Klinefelter syndrome (KS) is the most common genetic cause of human infertility, but the mechanism(s) responsible for its phenotype remain largely unknown. KS is associated with alterations in body composition and a high risk of developing metabolic diseases. We therefore hypothesized that KS men seeking fertility treatment possess an altered testicular metabolism profile that may hamper the nutritional support of spermatogenesis. Testicular biopsies from control (46, XY) (n = 6) and KS (47, XXY) (n = 6) individuals were collected and analyzed by proton high-resolution magic-angle spinning nuclear magnetic resonance spectroscopy. The mRNA and protein expression of crucial glycolysis-associated enzymes and transporters were evaluated in parallel by quantitative PCR and Western blot, respectively. Our data revealed altered regulation of glucose transporters (GLUT1 and GLUT3); phosphofructokinase 1, liver isoform (PFKL); and lactate dehydrogenase A (LDHA) expression in the testis of KS patients. Moreover, we detected a severe reduction in lactate and creatine accumulation within testicular tissue from KS men. The aberrant levels of the biomarkers detected in testicular biopsies of KS men may therefore be associated with the infertility phenotypes presented by these men. This article is protected by copyright. All rights reserved.
    Full-text · Article · Dec 2015 · Molecular Reproduction and Development
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    ABSTRACT: Ovarian tissue cryopreservation represents a valid strategy to preserve ovarian function in patients with a high risk of premature ovarian failure. We present a case of ovarian tissue cryopreservation carried out in an 18-year-old woman after a laparotomy for left adnexal mass with left adnexectomy. Congenital absence of the right ovary was observed during surgery. To preserve fertility, rescue cryopreservation of ovarian tissue was carried out under extreme conditions (without adopting the standard published protocol, not yet available at our centre). Ten years later, transplantation of cryopreserved ovarian tissue was carried out and, shortly after it, restoration of ovarian function was confirmed.
    No preview · Article · Dec 2015 · Reproductive biomedicine online
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    ABSTRACT: Introduction: Metformin is an oral antidiabetic agent used for treatment of patients with type 2 Diabetes mellitus. Recently, other applications for metformin have emerged, including the treatment of reproductive pathologies associated with hyperinsulinemia. Nonetheless, its effects on human sperm remain scarcely known. Commonly, sperm incubation decreases sperm function, compromising the success of the fertility treatment. Therefore, sperm preservation is crucial during preparation for Assisted Reproductive Technology treatments. Since metformin has been reported to possess antioxidant properties and act as a metabolic modulator, we hypothesized that it may be a good additive to sperm media during shortterm incubation. Methods: Human sperm obtained from 12 male patients were incubated for 2h at 37ºC in sperm preparation medium supplemented with increasing concentrations of metformin. Sub-pharmacological (5 µM), pharmacological (50 µM) and supra-pharmacological (500 µM) metformin concentrations were the evaluated doses in our research. At collection time and after incubation, sperm parameters and DNA integrity were evaluated. Sperm oxidative damages, such as protein carbonylation and nitration and lipid peroxidation were also quantified and metabolism was studied by proton nuclear magnetic resonance spectroscopy (1H-NMR). Results: Oxidative profile of human sperm was not affected after incubation with metformin. However, the pharmacological concentration of metformin decreased sperm quality, increasing sperm immotility and immature chromatin condensation. Additionally, it also increased sperm glycolytic flux. On the other hand, the supra-pharmacological concentration of metformin preserved sperm quality and DNA integrity. Conclusion: A supra-pharmacological metformin concentration was able to preserve sperm quality and DNA integrity of morphologically normal sperm. Therefore, at this concentration, metformin may be a beneficial supplement for sperm cryopreservation media.
    Full-text · Conference Paper · Sep 2015
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    ABSTRACT: Introduction: Pioglitazone is a potent synthetic agonist for the nuclear receptor peroxisome proliferator-activated receptor γ used, alone or in combination with metformin, to treat type 2 Diabetes mellitus (T2DM). In recent years, new clinical benefits have been described to pioglitazone but its effects on male reproductive system have not been investigated though other antidiabetic drugs, such as metformin, were reported to regulate the nutritional support of spermatogenesis by Sertoli cells (SCs). Thus, we propose to study human SCs (hSCs) metabolism after exposure to pioglitazone alone or in combination with metformin. We hypothesize that Pioglitazone can act as a modulator of SCs metabolism, altering the nutritional support of spermatogenesis. Methods: To test our hypothesis, hSCs were cultured in the presence of pioglitazone (1, 10, 100 μM) and pioglitazone 1.5 μM plus metformin (10 μM). hSCs were obtained from testicular biopsies from six men under treatment for recovery of male gametes and presenting anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis. Protein levels of glycolysis-related enzymes and transporters were determined by Western blot. Lactate dehydrogenase activity was spectrophotometrically assessed. Mitochondrial complexes levels were studied by Western blot and mitochondrial membrane potential was studied with a dye (JC-1). Finally, metabolite production and consumption were determined by 1H-NMR. Results: The suprapharmacological concentration of pioglitazone increased glucose consumption and mitochondrial complex II protein levels in hSCs though mitochondrial membrane potential was decreased. On the other hand, the pharmacological concentration of pioglitazone (10 μM) stimulated lactate production and allowed the establishment of important correlations among several key intervenient of glycolysis in hSCs. Treatment of hSCs with pioglitazone and metformin did not alter the glycolytic profile of these cells. Nevertheless, it induced alterations in the expression of mitochondrial complexes III and V in hSCs. Conclusions: Overall, our results provide clear evidence that the pharmacological dose of pioglitazone (10 μM) increases the efficiency of hSCs glycolytic flux. This led to an increase in lactate production, which is known to improve spermatogenesis. Thus, pioglitazone might be considered a suitable antidiabetic drug for men in reproductive age and with potential to improve male fertility.
    Full-text · Conference Paper · Sep 2015
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    ABSTRACT: Introduction: Dehydroepiandrosterone (DHEA) is a precursor of about 30-50% of androgens in adult men. DHEA action is partially exerted through its metabolites. Within these, 7-oxo-dehydroepiandrosterone (7-oxo-DHEA) is a biological DHEA metabolite that is not convertible to androgens and has proven to be a promising therapeutic agent. The process of spermatogenesis, central to male fertility, is strongly regulated by hormones, including androgens. Sertoli cells (SCs) constitute the main support of spermatogenesis. They present high metabolic rates to ensure lactate production for the developing germ cells, which may lead to high oxidative stress. Since any disruptions in androgen synthesis may compromise SCs function and male fertility, we aimed to evaluate the effects of DHEA and 7-oxo-DHEA in the human SCs (hSCs) metabolism and oxidative profile. Methods: hSCs were exposed to increasing concentrations of DHEA and 7-oxo-DHEA (0.025, 1 and 50 µM) that revealed to be non-cytotoxic in these experimental conditions. The consumption/production of metabolic substrates by hSCs were measured by 1H-NMR. Additionally, protein levels of key players of hSCs glycolytic pathway were evaluated by Western blot. The levels of protein oxidation and nitration, as well as lipid peroxidation were measured by Slot blot to evaluate hSCs oxidative status. Results: The obtained data demonstrated that 7-oxo-DHEA is a more potent metabolic modulator than DHEA since it increased hSCs glycolytic flux. DHEA seem to redirect hSCs metabolism to the Krebs cycle, while 7-oxo-DHEA has some inhibitory effect in this path. The highest 7-oxo-DHEA concentrations (1 and 50 µM) also increased lactate production, which is of extreme relevance for the successful progression of spermatogenesis in vivo. None of these steroids significantly altered the intracellular oxidative profile of hSCs, illustrating that at concentrations used they do not have pro- nor antioxidant actions in hSCs. Conclusions: Overall, we found that DHEA and 7-oxo-DHEA induce distinct alterations in hSCs metabolism, which transduce the respective androgenic and non-androgenic effects of these steroids. The exposure of hSCs to DHEA seems to stimulate the Krebs cycle, while exposure to 7-oxo-DHEA increased hSCs glycolytic flux. Our study represents a further step in the establishment of safe doses of DHEA and 7-oxo-DHEA to hSCs, supporting its possible use in hormonal and non-hormonal therapies against male reproductive problems.
    Full-text · Conference Paper · Sep 2015
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    ABSTRACT: Introduction: Klinefelter syndrome (KS) is characterized by X chromosome polysomy, with X disomy being the most recurrent variant. KS is frequently associated with hypogonadism, infertility and gynaecomastia. Adults with KS are characterized by severe hormonal deregulation, particularly hypergonadotropic hypogonadism with elevated serum concentrations of estradiol (E2). Estrogens play important roles in the regulation of testes development and spermatogenesis, through the interaction with their specific receptors. The expression of E2 receptors, namely G protein-coupled estrogen receptor (GPR30), estrogen receptor α (ERα) and estrogen receptor β (ERβ), has been described in male reproductive tract, but no data is available on the expression of these receptors in men with KS. Herein, we aimed to evaluate the mRNA expression of GPR30, ERα and ERβ in testis of men with KS as compared to those with conserved spermatogenesis and 46XY karyotype. Methods: Six human testicular biopsies were obtained from individuals with an average age of 44±4 years, confirmed 46XY karyotype, conserved spermatogenesis and seeking fertility treatment due to anejaculation, vasectomy or traumatic section of vas deferens (Control group). Other six testicular biopsies were obtained from azoospermic men with KS, average age of 34±3 years and confirmed homogeneous 47XXY karyotype (KS group). RNA was extracted from testicular tissue. cDNA was synthesized for each sample and gene expression of the three estrogen receptors (GPR30, ERα and ERβ) was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative PCR (RT-qPCR). Results: We were able to detect the presence of ERα, ERβ and GPR30 transcripts in the testicular tissue of individuals from Control and KS groups by RT-PCR. Our results show that ERβ transcripts are the most abundant in the testicular tissue of 46XY men. Furthermore, the testicular abundance of GPR30 mRNA in men with KS was approximately twelve times higher as compared to that of men with conserved spermatogenesis. Conclusions: GPR30 is to be an important mediator of E2 effects over steroidogenesis and may be partly responsible for the testicular alterations observed in men with KS. Hence, although the role of GPR30 in testes of KS men needs to be fully investigated, this E2 receptor may be a possible therapeutic target in these men in order to reduce the implications of the higher levels of E2 observed in those individuals.
    Full-text · Conference Paper · Sep 2015
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    ABSTRACT: Introduction: Male fertility can be disrupted by several chemotherapeutic drugs like etoposide. Etoposide is a topoisomerase II inhibitor that induces permanent double-stranded DNA breaks. It affects spermatogenesis and impairs fertility recovery after treatment. There is a need to identify a compound able to preserve male fertility during etoposide exposure. N-acetylcysteine (NAC) has chemopreventive and antioxidant properties. It has also been demonstrated that it is safe for clinical use and has several protective effects on cells, namely sperm from oxidative stress-induced damages. Due to its reported cytoprotector properties, we theorised that NAC may preserve sperm quality during etoposide exposure. Methods: Human sperm were incubated for 2h at 37 ºC with 25 µg/ml of etoposide, 50 µM of NAC and both drugs in combination. Conventional sperm parameters, DNA fragmentation and chromatin condensation were evaluated. Oxidative damages were measured and sperm metabolism was studied by proton nuclear magnetic resonance spectroscopy (1H-NMR). Results: Short-term exposure of sperm to etoposide induced chromatin alterations and DNA fragmentation but not sperm oxidative damages nor glycolytic profile alterations. The addition of NAC to sperm exposed to etoposide preserved sperm morphology and chromatin condensation and reduced sperm DNA fragmentation. Conclusion: Although short-term exposure to etoposide does not affect sperm vitality, it induces severe chromatin alterations and DNA damages. NAC addition to sperm exposed to etoposide protected chromatin integrity and reduced DNA fragmentation. NAC acts as a cytoprotector agent, shielding spermatozoa DNA from etoposide-induced damages. Exposure of sperm to etoposide does not induce cellular oxidative damages nor glycolytic profile alterations, providing evidence that etoposide directly affects DNA. NAC’s ability to preserve sperm DNA may be of clinical relevance, as the majority of patients undergoing chemotherapy fail to collect semen samples prior to treatment initiation. Thus, the integrity of the collected sperm after etoposide treatment initiation could be preserved with NAC addition, assuring that the majority of viable cells would have their DNA integrity preserved and could be safely used in assisted reproductive technologies.
    Full-text · Conference Paper · Sep 2015
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    ABSTRACT: The formation of competent spermatozoa is a complex event that depends on the establishment of adequate environments throughout the male reproductive tract, in which the maintenance of proper ionic contents in the luminal milieus plays a crucial role. HCO3- is essential not only to ionic homeostasis but also to pH maintenance along the male reproductive tract. Herein we determined the effect of E2 on the expression/functionality of sodium dependent HCO3- transporters from SLC4 family in human Sertoli cells (SCs). All the studied transporters (NBCn1, NBCe1 and NDCBE) were expressed in human SCs. We localized NBCn1 and NBCe1 on the basolateral portion of membrane of human SCs, while NDCBE was detected on the apical region of membrane of the polarized human SCs. Previous studies support an association of 17b-estradiol (E2) levels with modulation of specific ion transporters expression. In E2-treated human SCs (100nM) we could observe an increase in NBCn1, NBCe1 and NDCBE protein levels, as well as altered intracellular pH and transcellular transport. E2-treated SCs presented also a significant perturbation of ATP-induced short-circuit current and significant alterations on intracellular pH shift when DIDS the inhibitor. Overall, we report a relation between increased E2 levels and the expression/function of NBCn1, NBCe1 and NDCBE in human SCs, providing new evidence on the mechanisms by which E2 can regulate SCs physiology and consequently spermatogenesis, with direct influence on male reproductive potential.
    Full-text · Conference Paper · Sep 2015
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    ABSTRACT: Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in tea (Camellia sinensis (L.) Kuntze, Theaceae). Most of the medicinal properties attributed to tea are suggested to be due to EGCG. Herein, we evaluated the effect of EGCG (5 and 50 μM) on human Sertoli cells (hSCs) metabolism, mitochondrial functionality and oxidative profile. Exposure to 5 μM EGCG did not cause major alterations on hSCs, but 50 μM of EGCG induced a higher consumption of glucose and pyruvate to sustain the same production of lactate. In fact, hSCs exposed to 50 μM of EGCG presented a decrease in LDH and MCT4 levels, as well as LDH activity, suggesting that the efficiency of lactate production (essential for germ cell development) could be compromised. These metabolic alterations promoted by 50 μM EGCG on hSCs were accompanied by an increased lactate/alanine ratio, which is linked with the NAD+/NADH ratio. This alteration on cellular redox status might be associated with mitochondrial dysfunction observed in hSCs of this group. Interestingly, we verified that oxidative damage to proteins and lipids were decreased in hSCs exposed to 50 μM EGCG. This study suggests that EGCG is a metabolic modulator of hSCs but it appears to be safe to men in reproductive age. Interestingly, our study also illustrates that despite EGCG (50 μM) induces mitochondrial dysfunction in SCs, the oxidative damage is lower than is control cells. Since SCs are responsible for the nutritional support of spermatogenesis and present a Warburg-like metabolism, these findings suggest that EGCG may protect SCs from oxidative damage which may be relevant to male reproductive health.
    Full-text · Conference Paper · Aug 2015
  • M. Sousa · M. Gouveia · L. Ferraz · A. Barros · E. Oliveira · A. Alves · R. Sá

    No preview · Article · Aug 2015 · Microscopy and Microanalysis
  • Sara Bucar · Ana Gonçalves · Alberto Barros · Mário Sousa · Rosália Sá

    No preview · Article · Aug 2015 · Microscopy and Microanalysis
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    ABSTRACT: The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distribution were observed between ooplasm regions: cortical vesicles (C: 1.3% versus SC: 0.1%, IC: 0.1%, P = 0.001) and medium-sized vesicles containing zona pellucida-like materials (C: 0.2% versus SC: 0.02%, IC: 0%, P = 0.004) were mostly observed at the oocyte cortex, whereas mitochondria (C: 3.6% versus SC: 6.0%, IC: 7.2%, P = 0.005) were preferentially located in the subcortex and inner cytoplasm, and SER very large vesicles (IC: 10.1% versus C: 0.9%, SC: 1.67%, P = 0.001) in the oocyte inner cytoplasm. Further quantitative studies are needed in immature metaphase-I and mature metaphase-II oocytes, as well as analysis of correlations between ultrastructural and molecular data, to better understand human oocyte in vitro maturation.
    No preview · Article · Jul 2015 · Zygote
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    ABSTRACT: Caffeine is a widely consumed substance that has been reported to be a modulator of several cells metabolism. Since the metabolism of Sertoli cells (SCs) is essential for spermatogenesis, we aimed to study the effects of caffeine in human SCs (hSCs) metabolic pathways and hence in male reproductive health. hSCs were cultured in the absence or presence of caffeine (5, 50 and 500 µM) and its glycolytic profile was evaluated by studying glucose consumption and the production of lactate and alanine. Protein expression levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK1), lactate dehydrogenase (LDH) and monocarboxylate transporter 4 (MCT4) were determined, as well as LDH activity. Besides, caffeine has demonstrated some beneficial antioxidant effects, which led us to evaluate the antioxidant capacity of hSCs, the formation of carbonyl groups and lipid peroxidation. Caffeine at the lowest concentrations (5 and 50 µM) stimulated lactate production, but only hSCs exposed to 50 µM showed increased GLUTs expression. At the highest concentration (500 µM), LDH activity was stimulated to sustain lactate production. Notably, the antioxidant capacity of hSCs was decreased in a dose-dependent way and SCs exposed to 500 µM presented a pro-oxidant potential. hSCs exposed to 50 µM presented lower protein oxidation and lipid peroxidation. Moderate consumption of caffeine appears to be safe to male reproductive health since it stimulates lactate production by hSCs, which can promote germ cells survival. Nevertheless, caution should be taken with excessive caffeine consumption to avoid deleterious effects in hSCs functioning and thus, abnormal spermatogenesis.
    Full-text · Conference Paper · Jul 2015
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    ABSTRACT: In this prospective comparative study, sperm DNA fragmentation (sDNAfrag) was compared at each step of a sequential semen preparation, with semen parameters according to their degree of severity. At each step (fractions) of the sequential procedure, sDNAfrag was determined: fresh (Raw), after gradient centrifugation, washing, and swim-up (SU) for 70 infertile men enrolled in intracytoplasmic sperm injection cycles. sDNAfrag significantly (P = 0.04; P < 0.0001) decreased throughout the steps of semen preparation, with centrifugation and washing not increasing it. A negative correlation to sperm motility was observed in Raw and SU fractions, and a higher sDNAfrag was observed in samples with lower semen quality. Our results confirm that the steps of the sequential procedure do not compromise sperm DNA integrity and progressively decreased sDNAfrag regardless of the sperm abnormality and that semen parameters with lower quality present higher sDNAfrag. Four distinct patterns were observed, of which the entire sperm head staining was the pattern most expressed in all studied fractions. Additionally, the sperm head gene-rich region staining pattern was reduced by the procedure. This suggests that pattern quantification might be a useful adjunct when performing sDNAfrag testing for male infertility. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
    No preview · Article · Jul 2015 · Reproductive biomedicine online

Publication Stats

2k Citations
530.82 Total Impact Points

Institutions

  • 1995-2015
    • University of Porto
      • Faculty of Medicine
      Oporto, Porto, Portugal
  • 2009
    • Universidade da Beira Interior
      • Research Centre of Health Sciences (CICS)
      Ковильян, Castelo Branco, Portugal
    • Institut Marqués, Spain, Barcelona
      Barcino, Catalonia, Spain
  • 2001
    • Porto Military Hospital
      Oporto, Porto, Portugal