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ABSTRACT: Although low levels of reactive oxygen species (ROS) play a physiological role in maintaining sperm function, an increase in ROS generation above these levels may result in the induction of sperm membrane and DNA damage. The main objective of this study was to determine whether bovine oviducal explants (TU) and their conditioned media (CM) have a modulatory effect on the production of ROS, and consequently, on sperm DNA integrity. Thawed sperm were exposed to bovine TU and to CM obtained from the ampullar and isthmal regions after 4 and 12h, and DNA damage and intracellular ROS production was assessed by TUNEL and DHE and SYTOX Green, respectively. Co-incubation of spermatozoa with oviducal explants from the ampullar region (TUa) for 4h resulted in a statistically significant increase in the percentage of spermatozoa with DNA damage compared with controls (P=0.0106), and this increase was positively correlated with ROS levels. Conversely, although the incubation of spermatozoa with explants and conditioned media from the isthmal region (TUi and CMi, respectively) for 12h resulted in an increase of spermatozoa with DNA damage compared with controls (P<0.0001), this increase was not correlated with ROS levels. In conclusion, significant oxidative stress may take place in the oviduct, particularly during short-term incubation, and this may be related to changes in the antioxidant factors present in the oviducal cells and secretions. A redox imbalance in pro-oxidants and antioxidants in the oviduct may lead to oxidative stress and sperm DNA damage.
Reproduction Fertility and Development 08/2012; 24(6):871-6. · 2.11 Impact Factor
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ABSTRACT: Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins β3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain.
Cytometry Part A 01/2012; 81(1):65-71. · 3.73 Impact Factor
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ABSTRACT: alpha-Latrotoxin is the principal component of the venom from the euroasiatic Black Widow spider and has been studied for its pharmacological use as a synaptic modulator. Interestingly, smaller molecular weight fractions have been found to be associated with this toxin, but their cellular actions have not been studied in detail. The venom from the Chilean Black Widow spider (Latrodectus mactans) does not produce alpha-latrotoxin, however it does contain several small polypeptides. We have recently demonstrated cellular effects of these peptides at the synaptic level using whole-cell patch clamp techniques. Purified venom from the glands of L. mactans was studied in 12 DIV rat hippocampal neuronal cultures. Venom at a concentration of 10nM was able to decrease neuronal conductance thereby increasing membrane resistance. This effect on the passive properties of the neurons induced a change in action potential kinetics simulating the action of classic potassium channel blockers. These changes produced an increase in spontaneous synaptic activity in rat hippocampal cultures in the presence of the venom in a concentration- and time-dependent manner. These results indicate that venom from Chilean spider L. mactans is capable of increasing cell membrane resistance, prolonging the action potential and generating an increase in synaptic activity demonstrating an interesting pharmacological effect of these low molecular weight fragments.
NeuroToxicology 10/2008; 29(6):1121-6. · 3.10 Impact Factor
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ABSTRACT: A study was made of the effects of the venom of the Chilean spider Latrodectus mactans on endogenous ion-currents of Xenopus laevis oocytes. 1 microg/ml of the venom made the resting plasma membrane potential more negative in cells voltage-clamped at -60 mV. The effect was potentially due to the closure of one or several conductances that were investigated further. Thus, we determined the effects of the venom on the following endogenous ionic-currents: (a) voltage-activated potassium currents, (b) voltage-activated chloride-currents, and (c) calcium-dependent chloride-currents (Tout). The results suggest that the venom exerts its action mainly on a transient outward potassium-current that is probably mediated by a Kv channel homologous to shaker. Consistent with the electrophysiological evidence we detected the expression of the mRNA coding for xKv1.1 in the oocytes.
Biochemical and Biophysical Research Communications 09/2008; 375(4):571-5. · 2.48 Impact Factor
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ABSTRACT: To determine the effect of arachnotoxin (ATx), a venom extracted from the Chilean spider Latrodectus mactans, on intracellular calcium ([Ca(2+)](i)) and pH (pH(i)) in capacitated human spermatozoa.
Spermatozoa were collected from fertile adult men (n = 8). Mobile spermatozoa were collected by the "swim up" technique and stimulated with the crude extract of ATx and with progesterone (P).
Hospital of the Federal University of São Paulo, São Paulo, Brazil.
[Ca(2+)](i) was measured in fura2-AM-loaded spermatozoa, and pH(i) was measured in spermatozoa loaded with the pH-sensitive dye [(2',7')-bis (carboxymethyl)-(5,6)-carboxyfluorescein]-AM (BCECF).
The ATx and P induced a biphasic change in [Ca(2+)](i) consisting of a peak followed by a small but sustained elevation. The response to ATx was greatly reduced by pretreatment with P. The ATx caused intracellular acidification, whereas P induced alkalinization. Blockade of the NA(+)/H(+) exchanger with ethylisopropylamiloride (EIPA) sharply increased ATx-induced acidification.
Arachnotoxin increased [Ca(2+)](i) through the opening of calcium channels and release of calcium from intracellular stores. The ATx reduced pH(i) in human sperm, possibly by inhibiting the Na(+)/H(+) exchanger.
Fertility and sterility 07/2007; 87(6):1345-9. · 3.97 Impact Factor
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ABSTRACT: Ion channels have been assigned a pivotal importance in various sperm functions and are therefore promising targets for contraceptive development. The lack of data on channel functionality and pharmacology has hampered this goal. This is a consequence of technical problems of applying electrophysiological techniques to spermatozoa due to their small size and form. By using a laminin coating to increase adherence of spermatozoa and nystatin in the patch pipette for pore formation, we have adapted the whole-cell recording technique to study currents in mature uncapacitated bovine spermatozoa. Employing these conditions, in the head region, patched spermatozoa could be transferred into the whole-cell configuration. For the first time we document an outward rectifying current in mature bovine spermatozoa was blocked by tetraethyl ammonium (TEA) chloride. The observation of a shift in the reversal potential as a response to changes in the extracellular concentration of K(+) ions allowed us to identify this current as K(+) selective. This result shows that K(+) channels in the head region of mature uncapacitated bovine spermatozoa can be suitably investigated using the whole-cell recording patch-clamp technique.
Systems biology in reproductive medicine 54(6):231-9. · 0.80 Impact Factor
