In the last 40 years, male reproductive health—which is very sensitive to both environmental expo... more In the last 40 years, male reproductive health—which is very sensitive to both environmental exposure and metabolic status—has deteriorated and the poor sperm quality observed has been suggested to affect offspring development and its health in adult life. In this scenario, evidence now suggests that epigenetics shapes endocrine functions, linking genetics and environment. During fertilization, spermatozoa share with the oocyte their epigenome, along with their haploid genome, in order to orchestrate embryo development. The epigenetic signature of spermatozoa is the result of a dynamic modulation of the epigenetic marks occurring, firstly, in the testis—during germ cell progression—then, along the epididymis, where spermatozoa still receive molecules, conveyed by epididymosomes. Paternal lifestyle, including nutrition and exposure to hazardous substances, alters the phenotype of the next generations, through the remodeling of a sperm epigenetic blueprint that dynamically reacts to a...
The reduced number of animals in most wild felid populations implies a loss of genetic diversity.... more The reduced number of animals in most wild felid populations implies a loss of genetic diversity. The death of juveniles, prior to the production of mature sperm, represents a loss of potential genetic contribution to future populations. Since 2011 mouse testicular organ culture has introduced an alternative mechanism to produce sperm in vitro from immature tissue. However, extension of this technology to other species has remained limited. We have used the domestic cat (Felis catus) as a model for wild felids to investigate spermatogenesis initiation and regulation, with the mouse serving as a control species. Testicular tissue fragments were cultured in control medium or medium supplemented with knockout serum replacement (KSR), AlbuMax, beta-estradiol or AlbuMax plus beta-estradiol. Contrary to expectations, and unlike results obtained in mouse controls, no germ cell differentiation could be detected. The only germ cells observed after six weeks of culture were spermatogonia rega...
Molecular reproduction and development, Jan 21, 2017
Development of multicellular organisms is driven by a series of mitotic divisions after fertiliza... more Development of multicellular organisms is driven by a series of mitotic divisions after fertilization. Total cytoplasmic volume does not increase during this process, resulting in an increasing number of smaller daughter cells. Cell divisions are initially synchronous in an early embryo, but this ends at the 16-cell stage in the sea urchin. Orchesterated mitosis does, however, continue in subpopulations via the coordination of two different processes: karyokinesis and cytokinesis. Part of this asynchrony is due to the differentiation of lineages. Sea urchin (Lytechinus variegatus) gametes are readily obtained, and microinjection of their oocytes and eggs is easy (Carroll et al 1999, Dev Biol 206:232-247). Furthermore, the eggs and embryos are clear, allowing for the microscopic visualization of their rapid development, from the cleavage stages through blastula and eventually to larvae. An example of the orchestrated mitosis among a specific lineage of cells is observed in this Lytec...
Besides known factors that may cause male infertility, systemic diseases such as diabetes mellitu... more Besides known factors that may cause male infertility, systemic diseases such as diabetes mellitus may further exacerbate a decline in male fertility. This metabolic disease, clinically characterised by a hyperglycaemic phenotype, has devastating consequences in terms of human health, with reproductive dysfunction being one of the associated clinical complications. Nonetheless, the mechanisms responsible for such alterations are still poorly understood due to the multiplicity of factors involved in the induced pathophysiological changes. With this in mind, we focused on the main mediator of diabetes-associated alterations and performed an in vitro approach to address the effects of high glucose conditions on spermatogenesis, avoiding other confounding in vivo factors. Mouse (5 days post partum) testis fragments were cultured on agar gel stands at a gas–liquid interface with either 5, 25 or 50 mM D-glucose for 3 weeks. Stereological analysis revealed that high D-glucose levels increa...
Although no information exists regarding the levels of p,p&am... more Although no information exists regarding the levels of p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) on reproductive fluids of heavily exposed populations, they are possibly quite high given the serum levels reported so far. In these populations altered semen quality has been reported, although the direct effects of this DDT metabolite on crucial sperm parameters remain largely unexplored. With this in mind, a long-term in vitro incubation that better mimics the putative continuous exposure of spermatozoa to p,p'-DDE in the female reproductive tract in vivo was used. Before compromising sperm viability, continuous p,p'-DDE exposure remarkably decreased sperm motility, possibly due to the combined reduction in the proportion of sperm with high mitochondrial membrane potential and cellular ATP levels, all of which were clearly more affected at 50 and 100 μM p,p'-DDE. Moreover, 25 μM p,p'-DDE was also able to promote a decline in sperm with high MMP, however without significantly affecting motility. On the other hand, p,p'-DDE at the highest concentration strongly inhibited the process of capacitation following 24h of incubation. In conclusion, human sperm function is affected by continuous high p,p'-DDE exposure which may ultimately compromise male fertility. Given our previously findings that showed a non-regulated Ca(2+) entry in the presence of p,p'-DDE, we suggest that this organochlorine may promote mitochondrial Ca(2+) overload which may culminate in a general mitochondrial dysfunction and cellular ATP depletion, thus affecting sperm fertilizing potential. Our findings suggest a broader understanding of the non-genomic mechanism of p,p'-DDE action in human sperm.
Parabens are widely used as preservatives in many foods, cosmetics, toiletries, and pharmaceutica... more Parabens are widely used as preservatives in many foods, cosmetics, toiletries, and pharmaceuticals due to their relatively low toxicity profile and to a long history of safe use. Parabens are alkyl esters of phydroxybenzoic acid and typically include methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben, isopropylparaben and benzylparaben. These compounds are known to have a null or very weak estrogenic activity in estrogen receptor assays in vitro. In recent years, an increasing concern has emerged regarding possible adverse effects of chemicals in food and in cosmetics on human reproduction outcomes. In developed countries about 15% of human couples are affected by infertility, almost half of these cases attributed to men, through low sperm motility or/and sperm count. It is known that a significant number of cases of male infertility results from exposure to xenobiotics, and also that testis mitochondria are particularly affected by drug-induced toxicity. The present review discusses evidence that parabens may not be as safe as initially thought, and suggests that the interaction between parabens and mitochondrial function in the testis may be key in explaining the contribution of parabens for a decrease in reproductive potential.
Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. ... more Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrialsensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality.
Different aspects of sperm function can be monitored with a wide array of microscopy techniques, ... more Different aspects of sperm function can be monitored with a wide array of microscopy techniques, from simple optical microscopy to sophisticated fluorescence microscopy (epifluorescence and confocal), and the level of analysis may be adjusted and complemented according to ...
In the last 40 years, male reproductive health—which is very sensitive to both environmental expo... more In the last 40 years, male reproductive health—which is very sensitive to both environmental exposure and metabolic status—has deteriorated and the poor sperm quality observed has been suggested to affect offspring development and its health in adult life. In this scenario, evidence now suggests that epigenetics shapes endocrine functions, linking genetics and environment. During fertilization, spermatozoa share with the oocyte their epigenome, along with their haploid genome, in order to orchestrate embryo development. The epigenetic signature of spermatozoa is the result of a dynamic modulation of the epigenetic marks occurring, firstly, in the testis—during germ cell progression—then, along the epididymis, where spermatozoa still receive molecules, conveyed by epididymosomes. Paternal lifestyle, including nutrition and exposure to hazardous substances, alters the phenotype of the next generations, through the remodeling of a sperm epigenetic blueprint that dynamically reacts to a...
The reduced number of animals in most wild felid populations implies a loss of genetic diversity.... more The reduced number of animals in most wild felid populations implies a loss of genetic diversity. The death of juveniles, prior to the production of mature sperm, represents a loss of potential genetic contribution to future populations. Since 2011 mouse testicular organ culture has introduced an alternative mechanism to produce sperm in vitro from immature tissue. However, extension of this technology to other species has remained limited. We have used the domestic cat (Felis catus) as a model for wild felids to investigate spermatogenesis initiation and regulation, with the mouse serving as a control species. Testicular tissue fragments were cultured in control medium or medium supplemented with knockout serum replacement (KSR), AlbuMax, beta-estradiol or AlbuMax plus beta-estradiol. Contrary to expectations, and unlike results obtained in mouse controls, no germ cell differentiation could be detected. The only germ cells observed after six weeks of culture were spermatogonia rega...
Molecular reproduction and development, Jan 21, 2017
Development of multicellular organisms is driven by a series of mitotic divisions after fertiliza... more Development of multicellular organisms is driven by a series of mitotic divisions after fertilization. Total cytoplasmic volume does not increase during this process, resulting in an increasing number of smaller daughter cells. Cell divisions are initially synchronous in an early embryo, but this ends at the 16-cell stage in the sea urchin. Orchesterated mitosis does, however, continue in subpopulations via the coordination of two different processes: karyokinesis and cytokinesis. Part of this asynchrony is due to the differentiation of lineages. Sea urchin (Lytechinus variegatus) gametes are readily obtained, and microinjection of their oocytes and eggs is easy (Carroll et al 1999, Dev Biol 206:232-247). Furthermore, the eggs and embryos are clear, allowing for the microscopic visualization of their rapid development, from the cleavage stages through blastula and eventually to larvae. An example of the orchestrated mitosis among a specific lineage of cells is observed in this Lytec...
Besides known factors that may cause male infertility, systemic diseases such as diabetes mellitu... more Besides known factors that may cause male infertility, systemic diseases such as diabetes mellitus may further exacerbate a decline in male fertility. This metabolic disease, clinically characterised by a hyperglycaemic phenotype, has devastating consequences in terms of human health, with reproductive dysfunction being one of the associated clinical complications. Nonetheless, the mechanisms responsible for such alterations are still poorly understood due to the multiplicity of factors involved in the induced pathophysiological changes. With this in mind, we focused on the main mediator of diabetes-associated alterations and performed an in vitro approach to address the effects of high glucose conditions on spermatogenesis, avoiding other confounding in vivo factors. Mouse (5 days post partum) testis fragments were cultured on agar gel stands at a gas–liquid interface with either 5, 25 or 50 mM D-glucose for 3 weeks. Stereological analysis revealed that high D-glucose levels increa...
Although no information exists regarding the levels of p,p&am... more Although no information exists regarding the levels of p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) on reproductive fluids of heavily exposed populations, they are possibly quite high given the serum levels reported so far. In these populations altered semen quality has been reported, although the direct effects of this DDT metabolite on crucial sperm parameters remain largely unexplored. With this in mind, a long-term in vitro incubation that better mimics the putative continuous exposure of spermatozoa to p,p'-DDE in the female reproductive tract in vivo was used. Before compromising sperm viability, continuous p,p'-DDE exposure remarkably decreased sperm motility, possibly due to the combined reduction in the proportion of sperm with high mitochondrial membrane potential and cellular ATP levels, all of which were clearly more affected at 50 and 100 μM p,p'-DDE. Moreover, 25 μM p,p'-DDE was also able to promote a decline in sperm with high MMP, however without significantly affecting motility. On the other hand, p,p'-DDE at the highest concentration strongly inhibited the process of capacitation following 24h of incubation. In conclusion, human sperm function is affected by continuous high p,p'-DDE exposure which may ultimately compromise male fertility. Given our previously findings that showed a non-regulated Ca(2+) entry in the presence of p,p'-DDE, we suggest that this organochlorine may promote mitochondrial Ca(2+) overload which may culminate in a general mitochondrial dysfunction and cellular ATP depletion, thus affecting sperm fertilizing potential. Our findings suggest a broader understanding of the non-genomic mechanism of p,p'-DDE action in human sperm.
Parabens are widely used as preservatives in many foods, cosmetics, toiletries, and pharmaceutica... more Parabens are widely used as preservatives in many foods, cosmetics, toiletries, and pharmaceuticals due to their relatively low toxicity profile and to a long history of safe use. Parabens are alkyl esters of phydroxybenzoic acid and typically include methylparaben, ethylparaben, propylparaben, butylparaben, isobutylparaben, isopropylparaben and benzylparaben. These compounds are known to have a null or very weak estrogenic activity in estrogen receptor assays in vitro. In recent years, an increasing concern has emerged regarding possible adverse effects of chemicals in food and in cosmetics on human reproduction outcomes. In developed countries about 15% of human couples are affected by infertility, almost half of these cases attributed to men, through low sperm motility or/and sperm count. It is known that a significant number of cases of male infertility results from exposure to xenobiotics, and also that testis mitochondria are particularly affected by drug-induced toxicity. The present review discusses evidence that parabens may not be as safe as initially thought, and suggests that the interaction between parabens and mitochondrial function in the testis may be key in explaining the contribution of parabens for a decrease in reproductive potential.
Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. ... more Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrialsensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality.
Different aspects of sperm function can be monitored with a wide array of microscopy techniques, ... more Different aspects of sperm function can be monitored with a wide array of microscopy techniques, from simple optical microscopy to sophisticated fluorescence microscopy (epifluorescence and confocal), and the level of analysis may be adjusted and complemented according to ...
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Papers by Renata Tavares