With the omnipresence of new technologies extensively employing portable communication systems, the considerably increasing exposure to EMFs has raised concerns about how EMF exposure might impact human health and has warranted more consideration towards how to avoid or alleviate the associating hazardous effects. The present study investigated the adverse effects of EMF exposure on male fertility in rats. The study also evaluated the protective potential of exogenously administered spermine against EMF-induced testicular and reproductive aberrations.
In the present study, EMF exposure resulted in a deranged testicular oxidative status as manifested by the significantly increased level of the lipid peroxidation marker, MDA, as well as the significantly decreased activities of the antioxidant enzymes, catalase and GPx. In particular, GPx is the chief enzyme responsible for H2O2 elimination in the testis (Peltola et al., 1992). Therefore, its reduced activity might ultimately lead to an imbalance in the GSSG/GSH redox couple, and hence predispose testicular tissue and sperms to aggravated H2O2-induced damage. Mitigation of the EMF-induced oxidative aberrations in the EMF+SP group signifies a marked antioxidant capacity of spermine. Likewise, polyamines have been shown to act as reactive oxygen species (ROS) scavengers (Ha et al., 1998) and to mediate antioxidative defense by up-regulating antioxidant protein expression (Krüger et al., 2013).
The impaired oxidative status resulting from EMF exposure could underlie the immunohistochemically revealed increase in the testicular expression of caspase-3, the key executioner of apoptosis. Our findings reveal a notable anti-apoptotic effect of spermine, obviously by virtue of its potent antioxidant capacity, as verified by significantly lowering testicular caspase-3 expression in EMF+SP rats compared to untreated EMF-exposed rats. In context, spermine has shown anti-apoptotic potential against lead and/or gamma irradiation-induced hepatotoxicity (Abu-Khudir et al., 2017). Also, the observed protective effect of spermine against apoptosis is in accordance with a former report of enhanced susceptibility to apoptosis under conditions of polyamine depletion in intestinal epithelial cells (Li et al., 2001).
The present results of the comet assay reveal testicular DNA fragmentation in rats exposed to EMF, an effect which was attenuated by treatment with spermine. ROS overproduction results in a higher frequency of DNA single- and double-strand breaks and more DNA protein cross-linking (La Vignera et al., 2013). In line with the presently observed ameliorative effect of spermine against DNA damage, a number of studies have reported a protective effect of polyamines against radiation-induced DNA strand breaks (Spotheim-Maurizot et al., 1995), crosslinks to proteins (Chiu and Oleinick, 1998), and base degradation (Douki et al., 2000). Several mechanisms have been proposed to explain the radioprotection conferred by polyamines, including singlet oxygen quenching (Khan et al., 1992), hydroxyl radical scavenging (Spotheim-Maurizot et al., 1995) and the compaction of the DNA helix into tightly packaged structures, thereby reducing the accessibility of DNA to hydroxyl radicals (Douki et al., 2000; Spotheim-Maurizot et al., 1995).
The disrupted sperm count, viability, motility and morphology observed in the EMF-exposed rats is in agreement with a previous report by Agarwal and co-workers (2008). The deranged sperm parameters could be explained in light of the observed oxidative, apoptotic and DNA perturbations. A former study reported that radiofrequency electromagnetic radiation exposure for 2 h per day for 35 days caused elevation in semen ROS level and sperm MDA level along with a decline in sperm GPx activity in rats (Kesari et al., 2011). The high content of polyunsaturated fatty acids in the plasma membrane and the relative scarcity of ROS scavenging enzymes, owing to the virtual absence of cytoplasm, render mature spermatozoa particularly vulnerable to ROS attack resulting in lipid peroxidation (Opuwari and Henkel, 2016). Lipid peroxidation has been shown to increase membrane permeability, diminish motility, and cause other aberrations that weaken the fertilizing capacity in mammalian spermatozoa (Rao et al., 1989). Distortions in sperm head and midpiece were formerly reported in radiofrequency-exposed rats and were ascribed to the excessive generation of ROS in rats under mobile phone radiation exposure (Kesari et al., 2011). Excessive ROS overproduction elicited by the exposure to mobile phone radiation evokes sperm apoptosis through enhancing sperm caspase-3 activity in mobile phone radiofrequency-exposed rats (Kesari and Behari, 2012). ROS also adversely alter the biofunctional sperm profile, resulting in a higher count of spermatozoa with fragmented DNA that is inversely correlated with sperm count, motility, morphology and fertilization rate (Opuwari and Henkel, 2016).
Thus, the enhanced DNA fragmentation might also contribute to the impaired sperm profile. In the same context, exposure of human spermatozoa to radiofrequency electromagnetic radiation in the frequency range and power density of mobile phones instigated mitochondrial ROS generation resulting in enhanced DNA base adduct formation and eventually DNA fragmentation (Odacı and Özyılmaz, 2015). Compelling literature supports a vital role of polyamines in testicular development and spermatogenesis. Ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme of polyamine biosynthesis, exhibits stage- and cell-specific expression in rodent testis during spermatogenesis showing considerable elevation during the first spermatogenic wave (Alcivar et al., 1989; Kaipia et al., 1990). Spermine is essential for the completion of spermatogenesis since transgenic mice with an inactivating mutation of spermine synthase exhibit infertility and arrested spermatogenesis at the spermatogonia and primary spermatocyte stages (Wang et al., 2004). The mechanisms underlying the ameliorative effect of polyamines on sperm motility are not fully elucidated, but in vitro studies provide a number of possible explanations linked to intracellular signaling and sperm energy metabolism. Spermine enhances glycolysis in rat epididymal spermatozoa (Pulkkinen et al., 1975), stimulates adenylate cyclase activity and decreases the activity of phosphodiesterase, an inhibitor of cAMP signaling in human spermatozoa (Shah et al., 1975).
As a marker for differentiating spermatogonia (Schrans-Stassen et al., 1999), c-kit down-regulation in the testicular tissue of EMF-exposed rats could indicate disruption of spermatogenesis, as also manifested by the observed perturbations in sperm count, viability, motility and morphology. The observed decline in c-kit mRNA level along with impaired spermatogenesis are in line with an earlier report (Shahin and Mohamed, 2017). The beneficial effect of spermine against EMF-associated defective spermatogenesis is obviously revealed by the markedly up-regulated gene expression level of c-kit in the EMF+SP group compared to the corresponding non-treated EMF group.
Exposure to EMF radiation resulted in significant activation of testicular NF-κB, as evidenced by the immunohistochemically detected overexpression of the NF-κB p65 subunit. This EMF-induced NF-κB activation could be explained on the basis that exposure to EMF alters the activities of protein kinase C and protein tyrosine kinases (Holian et al., 1996; Uckun et al., 1995) which, in turn, play key roles in the regulation of NF-κB (Baldwin, 1996; Imbert et al., 1996). As a key transcriptional activator of a variety of inflammatory genes, the activated NF-κB noted in the EMF group could explain the immunohistochemically detected overexpression of COX-2 and iNOS. The current work depicts a potent anti-inflammatory effect of spermine against EMF-induced testicular aberrations, as verified by its notable inhibitory effect on the activation of NF-κB and on the expression of its downstream pro-inflammatory effectors, COX-2 and iNOS. The suppressive effect of spermine on NF-κB p65 and COX-2 expression is supported by reports of induction of NF-κB activation and COX-2 up-regulation following polyamine depletion (Li et al., 2001; Parker and Gerner, 2002). The inhibitory effect of spermine on iNOS expression was formerly described (Blachier et al., 1997).
The present findings depict a notably altered serum hormone profile in the exposed group. A similar pattern of decline in serum inhibin B and testosterone levels along with elevation in FSH and LH levels was reported upon long term exposure of male rats to EMF (Odacı and Özyılmaz, 2015). The markedly reduced testosterone level in EMF-exposed rats is obviously the result of, firstly, their down-regulated testicular StAR, which mediates the rate-limiting step in steroidogenesis, and, secondly, their lowered testicular activities of 3β-HSD and 17β-HSD, the key enzymes in testicular androgenesis.
Noteworthy, activated NF-κB has been reported to down-regulate steroidogenic enzyme gene expression in Leydig cells (Hong et al., 2004), lending support to the currently observed pattern of alterations in NF-κB p65, StAR, 3β-HSD, 17β-HSD and testosterone. In view of the aforementioned justifications, the ability of spermine to boost steroidogenesis and to reinstate the testosterone level could be attributed to its ability to reverse the EMF-induced NF-κB activation. The restoration of normal testosterone level achieved by spermine treatment is consistent with the reported role of polyamines in steroidogenesis. It has been demonstrated that inhibition of ODC1 reduced steroid synthesis in rodent testis, suggesting a polyamine indispensability for steroidogenesis (Mäkitie et al., 2009).
The elevation of serum FSH level observed in EMF-exposed rats is considered an endocrine index of germinal epithelial damage (Bergmann et al., 1994), which is consistent with our histomorphometric observations. The observed elevations in serum LH and FSH levels of EMF-exposed rats are in accordance with a former report (Sepehrimanesh et al., 2014), and could possibly be the result of a feedback mechanism in response to the notable decline in the testosterone level (Shahin and Mohamed, 2017). Another important feedback signal for the production of FSH is the Sertoli cell hormone, inhibin B, which showed a significantly decreased serum level in EMF-exposed rats, thereby providing another explanation for the elevated serum level of FSH observed in these rats. As an important endocrine marker of spermatogenesis (Foppiani et al., 1999), inhibin B decline indicates impaired spermatogenesis which is in parallel with the observed decline in sperm parameters and testicular c-kit expression. Accordingly, the observed changes in serum testosterone level could account for the corresponding alterations in sperm parameters.
Seminiferous tubule epithelial height is an index of normal epithelial cell architecture with harmonic germinal cell associations. The decrease in epithelial height observed in exposed rats indicates defective cell connections in the germ epithelium, and could be attributed to the observed sloughing of cells in the exposed rat testis (A D et al., 2012). STBM seems to play a distinct role in spermatogenesis. The STBM serves as the proliferation site for spermatogonia, a support for the phenotypic differentiation of Sertoli cells, and for maintaining the immunological function of the seminiferous epithelium. In fact, ultrastructural anomalies in the STBM are assumed to cause infertility (Kahsai et al., 1997). Therefore, the defective sperm profile observed following EMF exposure could be related to the abnormally thickened STBM, an assumption that is further supported by the amended sperm parameters associating the restored STBM thickness in the spermine-treated group. Sufficient levels of testosterone are crucial for maintaining structural morphology and physiology of the seminiferous tubules and for proper functioning of the seminiferous epithelium (Holdcraft and Braun, 2004). Thus, the observed morphometric alterations resulting from EMF exposure and their effective reinstatement by spermine could be probably explained, at least in part, by the corresponding changes in testosterone levels.
In conclusion, the present study highlights the hazardous impact of EMF radiation exposure on male fertility in rats, as manifested by the impaired spermatogenesis and steroidogenesis and the disrupted sperm and hormone profiles. The study also substantiates the potent protective effect afforded by spermine against EMF-induced aberrations, which is probably mediated, at least in part, by amending EMF-associated oxidative, inflammatory, apoptotic and DNA perturbations.
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