The transverse oviduct sections performed on UTJ, I, AIJ, and A obtained from all experimental conditions, stained with HE and HE-AB, showed a normal histological morphology. In fact, all regional histological variations were confirmed: the tube wall thickened progressively toward the uterus, whereas the lumen diminished in size in this direction. In particular, the tunica mucosa of A and AIJ, in which fertilization usually occurs, was highly developed into characteristic longitudinal folds (plicae) which branched into a labyrinth of spaces (Fig. 5). On the contrary in the I, and UTJ the folds were low (data not shown). The lamina epithelialis was pseudostratified intermittently ciliated columnar which is typical of the pig, and showed normal-shaped cells with a regular morphology (Fig. 5). Moreover, the HE-AB, a specific stain for acid proteoglycans, allowed both to reveal the connective tissue of the lamina propria, and the secretory cells where it was possible to observe the presence of this kind of molecule in their cytoplasm, mainly near their luminal surface (Fig. 5). Interestingly, this aspect was evident also in the treated samples, assuming that the secretory activity of ELF-EMF exposed oviducts was not affected.
Finally, it was also possible to note in some tissue sections obtained from explanted oviduct after 12 h the presence of spermatozoa in the middle of the lumen of AIJ (Fig. 5) independently of the treatment considered.
4. Discussion
Despite the large number of epidemiological and experimental determinations carried out until now, the real effect of the ELF-EMF exposure on human (and mammalian) reproductive outcome is still unknown. Recently the possibility that these radiations can interfere with the function of male gamete has been proposed by our group, referring to an in vitro animal model [37]. In particular it was found that the mature boar spermatozoa exposure to a sinusoidal field of 50 Hz frequency and 1 mT intensity for 1-4 h, incubated under capacitating conditions, affected the acquisition of the fertilizing ability, as assessed by an IVF experiment. These findings opened a new perspective in the understanding of the interaction between male germ cells and ELF-EMF but, at the same time, posed some important questions: does the field effect follow a dose-dependent law? Is it possible to define a safety threshold? Which sperm population (i.e. uncapacitated or capacitated cells) is the target of the field effect? The ELF-EMF effects found in vitro are maintained in an in vivo model?
To answer these questions the present experimental set-up has been designed. In particular, at first, the relationship between the field intensity and the effects exerted on the sperm cells by the ELF-EMF exposure was investigated. The loss of acrosome integrity was chosen as a marker of cellular damage for several reasons: this parameter has been described to be sensitively and reliably related to the ELF-EMF exposure, its experimental determination can be easily carried out by a simple fluorescent stain, a spermatozoon with a damaged acrosome loses its fertilizing ability [37].
The result that the field intensity and the acrosomal loss are linked by a sigmoid law, is very remarkable. This datum allowed the definition of the sensitivity threshold of the system and the maximum effect that can be exerted by radiations. Until now the studies on biological systems sensitivity to the effect of ELF-EMF are completely insufficient for what concerns the reproductive sphere. These findings suggests that is possible to experimentally define the field intensity levels under which the exposure does not exert a specific negative effect. The definition of a maximum plateau in the cell damages induced by ELF-EMF (i.e. the saturation effect) is important because concurs to sustain the hypothesis that not all the spermatozoa are susceptible to be affected by ELF-EMF but only a subpopulation is the target. In fact, during the capacitation process, both in vitro and in vivo, many sperm subpopulatons, characterized by a specific pattern of emergence, coexist. Two different evaluations of fertilizing ability of exposed spermatozoa were carried out to test if the population, undergoing AR in response to ELF-EMF, has a specific physiological meaning. In the first one it was found that the response of treated spermatozoa to the sZP co-incubation was reduced depending on the field intensity. The pattern of the dependence of the two variables (the threshold of system response, the DT50 and the saturation level) was closely similar to that recorded for AR. This evidence allows to hypothesize that the spermatozoa population targeted by field effect is that of capacitating cells. More in particular resulted evident that when the field effect were below the threshold, the sZP responding ability was the maximum. Vice versa when the ELF-EMF induced damages reached the plateau the response to sZP was virtually absent. To complete the investigation the exposed spermatozoa were tested in an IVF trial. In this case also two important findings were confirmed: the dose dependence of the investigated phenomenon and the marked reduction in the fertilizing ability caused by the highest intensity field in terms of percentage of fertilized oocytes, in polyspermy rate and in mean number of spermatozoa/polyspermic oocyte. On these basis it is possible to speculate that the spermatozoa affected by the fields effect are those undergoing capacitation and that, as a consequence, the sperm cells fertilizing ability is negatively affected by the exposure to a filed of an intensity ≥0.75 mT. This finding is in agreement with the data previously reported [37] and can be explained considering that the ELF-EMF posses a very low energy content and, as a consequence, they are unable to determine direct molecular damages (such as ionization) but, as it has been hypothesized, they can act perturbing the cellular environment in a complex way. Panagopoulos et al. [48] and [49] suggested that the forced coherent vibration superimposed at ions by ELF-EMF could be able to irregularly gate electrosensitive channels. In addition, it has been developed an explanatory model considering the membranes and many other subcellular structures (cytoskeleton, biological polymers, proteins) as polyphasic liquid crystals. Indeed, a large literature exists on the ability of the magnetic fields to orientate liquid crystals and to form orientational and flow domains [50] and [51]: these perturbations in the architecture of cellular components could influence their function, as described in ELF-EMF exposed artificial systems [52]. The identification of ELF-EMF target and mechanism of action is a topic of relevant interest, in fact, it will make possible to design and to validate prophylactic or therapeutic strategies.
To validate the data obtained by in vitro experiments the investigation has been transposed in an in vivo model. In fact on one hand the magnetic field penetrates the biological structures without interference (as it happens during magnetic resonance imaging, MRI, procedures) and as a consequence the physical parameters (frequency, intensity, waveform) of the applied radiation in vitro and in vivo were identical. On the other hand, it is possible to speculate that the multicellular context, characterized by the interaction of male germ cells with the female structures, could repair or propagate the ELF-EMF effect on spermatozoa. To further increase the predictive value of data two different determinations were performed: the oviducts were exposed in the presence or without the spermatozoa, to separate the effects exerted by the fields on the two different components of the system (the male gametes and the female genital tract). The findings indicate two different kinds of events. Firstly at the highest field intensity (1 mT) it was evident a decrease in the fertilizing ability of spermatozoa, in fact the fertilization rate was approximately 30% less that that obtained in control conditions. This datum confirms the detrimental effect of ELF-EMF exposure on the functional asset of sperm cells, but shows a different system threshold. In vitro, at 0.75 mT intensity, it was evident a negative effect while in vivo the field started to induce alteration only at 1 mT. Moreover the exposure of the oviduct alone, at field intensities ≥0.75 mT, caused a decrease in the system performance as documented by the significative reduction of the percentage of zygotes of about 20% and of the fertilization rate of about 10%.
In absence of any evidence of morphological alterations induced by ELF-EMF exposure (as assessed by the histological evaluation) on the oviducts it was possible to explain this finding hypothesizing a functional damage. In the uterine tubes many events related to fertility take place and an alteration of the oviductal milieu could result in the system failure. For example an alteration of ciliar motility or of tubaric fluid flow could determine the fertilization reduction/retardation [53], [54], [55], [56] and [57]. Also the disequilibrium of fertilization related molecules secretion could promote the same effect. For instance many reports [57], [58] and [59] indicates that the porcine oviduct, under the stimulation of ovarian steroid hormones, de novo synthesizes the pig oviductal-specific secretory glycoprotein (pOSP) family into the lumen. The synthesis of these proteins into the oviductal lumen creates a microenvironment able to support important reproductive events, which include fertilization and early cleavage-stage embryonic development. At the same time it exists an intense cross-talking between the oviducts and the embryo in terms of growth factors and regulating molecules driving the embryo development [60]. An alteration of tubal environment and a delay in fertilization-related event could explain also the reduced number of blastocycts and the lo
The transverse oviduct sections performed on UTJ, I, AIJ, and A obtained from all experimental conditions, stained with HE and HE-AB, showed a normal histological morphology. In fact, all regional histological variations were confirmed: the tube wall thickened progressively toward the uterus, whereas the lumen diminished in size in this direction. In particular, the tunica mucosa of A and AIJ, in which fertilization usually occurs, was highly developed into characteristic longitudinal folds (plicae) which branched into a labyrinth of spaces (Fig. 5). On the contrary in the I, and UTJ the folds were low (data not shown). The lamina epithelialis was pseudostratified intermittently ciliated columnar which is typical of the pig, and showed normal-shaped cells with a regular morphology (Fig. 5). Moreover, the HE-AB, a specific stain for acid proteoglycans, allowed both to reveal the connective tissue of the lamina propria, and the secretory cells where it was possible to observe the presence of this kind of molecule in their cytoplasm, mainly near their luminal surface (Fig. 5). Interestingly, this aspect was evident also in the treated samples, assuming that the secretory activity of ELF-EMF exposed oviducts was not affected.
Finally, it was also possible to note in some tissue sections obtained from explanted oviduct after 12 h the presence of spermatozoa in the middle of the lumen of AIJ (Fig. 5) independently of the treatment considered.
4. Discussion
Despite the large number of epidemiological and experimental determinations carried out until now, the real effect of the ELF-EMF exposure on human (and mammalian) reproductive outcome is still unknown. Recently the possibility that these radiations can interfere with the function of male gamete has been proposed by our group, referring to an in vitro animal model [37]. In particular it was found that the mature boar spermatozoa exposure to a sinusoidal field of 50 Hz frequency and 1 mT intensity for 1-4 h, incubated under capacitating conditions, affected the acquisition of the fertilizing ability, as assessed by an IVF experiment. These findings opened a new perspective in the understanding of the interaction between male germ cells and ELF-EMF but, at the same time, posed some important questions: does the field effect follow a dose-dependent law? Is it possible to define a safety threshold? Which sperm population (i.e. uncapacitated or capacitated cells) is the target of the field effect? The ELF-EMF effects found in vitro are maintained in an in vivo model?
To answer these questions the present experimental set-up has been designed. In particular, at first, the relationship between the field intensity and the effects exerted on the sperm cells by the ELF-EMF exposure was investigated. The loss of acrosome integrity was chosen as a marker of cellular damage for several reasons: this parameter has been described to be sensitively and reliably related to the ELF-EMF exposure, its experimental determination can be easily carried out by a simple fluorescent stain, a spermatozoon with a damaged acrosome loses its fertilizing ability [37].
The result that the field intensity and the acrosomal loss are linked by a sigmoid law, is very remarkable. This datum allowed the definition of the sensitivity threshold of the system and the maximum effect that can be exerted by radiations. Until now the studies on biological systems sensitivity to the effect of ELF-EMF are completely insufficient for what concerns the reproductive sphere. These findings suggests that is possible to experimentally define the field intensity levels under which the exposure does not exert a specific negative effect. The definition of a maximum plateau in the cell damages induced by ELF-EMF (i.e. the saturation effect) is important because concurs to sustain the hypothesis that not all the spermatozoa are susceptible to be affected by ELF-EMF but only a subpopulation is the target. In fact, during the capacitation process, both in vitro and in vivo, many sperm subpopulatons, characterized by a specific pattern of emergence, coexist. Two different evaluations of fertilizing ability of exposed spermatozoa were carried out to test if the population, undergoing AR in response to ELF-EMF, has a specific physiological meaning. In the first one it was found that the response of treated spermatozoa to the sZP co-incubation was reduced depending on the field intensity. The pattern of the dependence of the two variables (the threshold of system response, the DT50 and the saturation level) was closely similar to that recorded for AR. This evidence allows to hypothesize that the spermatozoa population targeted by field effect is that of capacitating cells. More in particular resulted evident that when the field effect were below the threshold, the sZP responding ability was the maximum. Vice versa when the ELF-EMF induced damages reached the plateau the response to sZP was virtually absent. To complete the investigation the exposed spermatozoa were tested in an IVF trial. In this case also two important findings were confirmed: the dose dependence of the investigated phenomenon and the marked reduction in the fertilizing ability caused by the highest intensity field in terms of percentage of fertilized oocytes, in polyspermy rate and in mean number of spermatozoa/polyspermic oocyte. On these basis it is possible to speculate that the spermatozoa affected by the fields effect are those undergoing capacitation and that, as a consequence, the sperm cells fertilizing ability is negatively affected by the exposure to a filed of an intensity ≥0.75 mT. This finding is in agreement with the data previously reported [37] and can be explained considering that the ELF-EMF posses a very low energy content and, as a consequence, they are unable to determine direct molecular damages (such as ionization) but, as it has been hypothesized, they can act perturbing the cellular environment in a complex way. Panagopoulos et al. [48] and [49] suggested that the forced coherent vibration superimposed at ions by ELF-EMF could be able to irregularly gate electrosensitive channels. In addition, it has been developed an explanatory model considering the membranes and many other subcellular structures (cytoskeleton, biological polymers, proteins) as polyphasic liquid crystals. Indeed, a large literature exists on the ability of the magnetic fields to orientate liquid crystals and to form orientational and flow domains [50] and [51]: these perturbations in the architecture of cellular components could influence their function, as described in ELF-EMF exposed artificial systems [52]. The identification of ELF-EMF target and mechanism of action is a topic of relevant interest, in fact, it will make possible to design and to validate prophylactic or therapeutic strategies.
To validate the data obtained by in vitro experiments the investigation has been transposed in an in vivo model. In fact on one hand the magnetic field penetrates the biological structures without interference (as it happens during magnetic resonance imaging, MRI, procedures) and as a consequence the physical parameters (frequency, intensity, waveform) of the applied radiation in vitro and in vivo were identical. On the other hand, it is possible to speculate that the multicellular context, characterized by the interaction of male germ cells with the female structures, could repair or propagate the ELF-EMF effect on spermatozoa. To further increase the predictive value of data two different determinations were performed: the oviducts were exposed in the presence or without the spermatozoa, to separate the effects exerted by the fields on the two different components of the system (the male gametes and the female genital tract). The findings indicate two different kinds of events. Firstly at the highest field intensity (1 mT) it was evident a decrease in the fertilizing ability of spermatozoa, in fact the fertilization rate was approximately 30% less that that obtained in control conditions. This datum confirms the detrimental effect of ELF-EMF exposure on the functional asset of sperm cells, but shows a different system threshold. In vitro, at 0.75 mT intensity, it was evident a negative effect while in vivo the field started to induce alteration only at 1 mT. Moreover the exposure of the oviduct alone, at field intensities ≥0.75 mT, caused a decrease in the system performance as documented by the significative reduction of the percentage of zygotes of about 20% and of the fertilization rate of about 10%.
In absence of any evidence of morphological alterations induced by ELF-EMF exposure (as assessed by the histological evaluation) on the oviducts it was possible to explain this finding hypothesizing a functional damage. In the uterine tubes many events related to fertility take place and an alteration of the oviductal milieu could result in the system failure. For example an alteration of ciliar motility or of tubaric fluid flow could determine the fertilization reduction/retardation [53], [54], [55], [56] and [57]. Also the disequilibrium of fertilization related molecules secretion could promote the same effect. For instance many reports [57], [58] and [59] indicates that the porcine oviduct, under the stimulation of ovarian steroid hormones, de novo synthesizes the pig oviductal-specific secretory glycoprotein (pOSP) family into the lumen. The synthesis of these proteins into the oviductal lumen creates a microenvironment able to support important reproductive events, which include fertilization and early cleavage-stage embryonic development. At the same time it exists an intense cross-talking between the oviducts and the embryo in terms of growth factors and regulating molecules driving the embryo development [60]. An alteration of tubal environment and a delay in fertilization-related event could explain also the reduced number of blastocycts and the lo
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