**4.1 EMF-induced morphological changes and apoptosis in ovary**

The result of present study showed that EMF-exposure increased degenerative changes in the ovarian follicles. The results obtained from TEM studies have revealed that oocytes became shrunken, and the zona pellucida appeared narrower in the EMF exposed group in comparison to control group (Figs 1, 3, 4, 7 ). It was also shown that the number of microvilli in oocytes and coronal cells were decreased in experimental group. It is known that the microvilli of oocytes and granulosa cells are in contact, within the zona pellucida, by gap junction and are involved in oocyte nutrition (Martin et al. 2001; Takeo and Hokano 1995; Gondos 1982; Vazquez and Sotello 1967). The alterations produced by EMF could either be the result of initiation of apoptosis in the follicular cells or as a result of apoptosis in the oocytes themselves.

Fig. 25. Photomicrograph from uterine tube in EMF-exposed rat. Note apoptotic cells with golden brown color at luminal face (arrow). TUNEL method, counterstained with toluidine

This section is going to deal : 1) The morphological effects of EMF exposure on the genital organs including ovary, uterus, and Fallopian tubes using light and electron microscopy. 2) A possible role of apoptosis in the mediation of EMF-induced alterations using immunohistochemical techniques. To make it easier to explain, each organ is discussed

The result of present study showed that EMF-exposure increased degenerative changes in the ovarian follicles. The results obtained from TEM studies have revealed that oocytes became shrunken, and the zona pellucida appeared narrower in the EMF exposed group in comparison to control group (Figs 1, 3, 4, 7 ). It was also shown that the number of microvilli in oocytes and coronal cells were decreased in experimental group. It is known that the microvilli of oocytes and granulosa cells are in contact, within the zona pellucida, by gap junction and are involved in oocyte nutrition (Martin et al. 2001; Takeo and Hokano 1995; Gondos 1982; Vazquez and Sotello 1967). The alterations produced by EMF could either be the result of initiation of apoptosis in the follicular cells or as a result of apoptosis in the

separately, following the above theme, and made a general conclusion at the end.

**4.1 EMF-induced morphological changes and apoptosis in ovary** 

blue. 360X.

**4. Discussion** 

oocytes themselves.

Comparison of oocyte degeneration with apoptosis since Wyllie et al. (Wyllie 1980) first described the morphological characteristics of physiological cell death (apoptosis) few studies have described the ultrastructure of the atretic oocyte and none have examined this in pubertal or adult animals. Most reviews of ovarian follicular atresia focused on changes in granulosa cells or equate the entire process with apoptosis (Tilly 1998; Kaipia and Hsueh 1996). Biochemical analysis of atretic follicles that have measured DNA integrity (188-189)or increases in cell death – related mRNA levels, including bax and Fas, fasL (Hsuhe et al. 1994; Mori et al. 1997), have confirmed that apoptosis is occurring in antral ovarian follicles. Because the oocyts is a very small component of these large follicles, such measurements most probably reflect the status of granulosa cells. Due to these restrictions, microscopic examination is required to study the process of atresia in oocytes in situ. Because ultrastructural characterization is a reliable method for the classification of cell death as apoptosis (Payne et al. 1995).

Alterations in oocytes from EMF-exposed ovaries mainly in rat atretic f'ollicles included: loss of both granulosa cell and oocyte microvilli from the zona pellucida, changes in cytoplasmic organelles such as lamellar condensation and shirinkage of oocytes. Loss of microvilli and cytoplasmic condensation do resemble apoptosis, but other events differed from those associated with traditional apoptosis. For example, the mitochondria do not maintain their characteristic appearance during, early stages of atresia, as normally occur in apoptosis. While, cytoplasmic condensation, which is reflected by an increase in electron density at the ultrastructural level, observed in degenerating oocytes. In support of the findings of the present study, condensed chromatin was never observed in oocytes of atretic follicles by other investigators (Devine 2000). These comparisons suggest that there are more differences than similarities between physiological oocyte cell death and apoptosis. Other reports attempting to identify the mechanism of oocyte death have not discussed the possibility of alternative, nonapoptotic, types of physiological death (Perez et al. 1999). Early ultrastructural studies occurred before apoptosis was characterized (Vazquez and Sotello 1967; Franchi and Mandl 1962). More recent studies using ovulated oocytes failed to prove definitively that apoptosis was the mechanism of oocyte death (Perez et al. 1999; Van et al. 1998; Phillips et al. 1992). Therefore, it seems likely that oocytes in postnatal rats have unique cell-death triggers, signal transduction pathways, and clearance mechanisms as compared with other cell types. Such flexibility has not been described for traditional apoptosis.

#### **4.2 The unique aspects of oocytes**

The unique nature of the oocyte relative to other cell types may be the cause for its unusual manner of cell death. Oocytes can remain arrested in meiosis for years, are surrounded by an acellular zona pellucida, are nonproliferating, and are known to rely on surrounding granulosa cells for survival (Hirshfield 1991). Apoptosis is an active process thought to protect the rest of an organism from an aberrant cell. Meiotic oocyte may not be required to undergo apoptosis, because they pose no threat of excessive proliferation and tumor formation. Overall, the results presented here support that oocyte loss in atretic follicles of postnatal rats can be morphologically distinguished from the two more widely described mechanisms of cell death, necrosis, and apoptosis. While it is generally accepted that granulosa cells are lost by apoptosis, the ability of the oocyte to undergo apoptosis is still in

Environmental Electromagnetic Field and Female Fertility 79

peroxide (Sugino et al. 1996); and in addition, macrophage-derived cytokines such as transforming growth factor and have been found to induce apoptosis in ovarian cells (Foghi et al. 1996). The present study describe not only the ultrastracture of follicle in rat ovaries but also the increased degenerative changes within follicle committed to undergo apoptosis after EMFexposure (Roshangar and Soleimani Rad, 2007). In support of this idea several studies reported (Roshangar and Soleimani Rad 2001; Kaipia et al. 1997; Hughes and Gorospe 1991; Zamboni 1972) increased of macrophages after the EMF exposure. It is believed that the cell fragments produced by apoptosis are phagocytosed by macrophages. These macrophages do not release cytokines that would initiate an inflammatory response (Andrew et al. 1998).Thus the granulosa and thecal cells would be destroyed, leaving only fibroblasts and other connective tissue elements to represent the follicular wall, thereby transforming the follicle into a cystic follicle. It has been reported that macrophages were involved in the apoptotic process (Leonardo and Skeel 1980). This clearly indicates that increased number of macrophages in experimental group (Soleimani Rad and Roshangar 2000) potentially increases cell damages. Using rat ovaries, the present study confirms that the follicular granulosa cells undergo apoptosis after the EMF exposure on the basis of their microscopic features, i.e., condensation of nuclear chromatin to the margin of the nucleus, and presence of apoptotic bodies, both changes being characteristic of apoptosis (Ker et al. 1972), even more evidence is occurring in parallel with the above- mentioned morphological changes. As far as we aware, the fate of the

In the present study in rats, we demonstrated that large amount apoptotic and internalized granulosa cells and their fragments. The factors responsible for this basic difference are as yet unknown. In summary, this model is proposed to foster and more clearly focus future

Our results demonstrate that a remarkable proportion of oocytes in the rat ovary degenerate during the EMF exposure by the mechanism of apoptosis. This is already evident at experimental period, with a high number of apoptotic oocytes and increasing of macrophages and autophagic vacuoles in some occasional granulosa cells, and several lipid droplets in thecal and luteal cells. Previous TEM studies (Takeo and Hokano 1995) suggested that the process of apoptosis of the ingested cells was assumed to progress through the following steps. The nuclei of ingested cells underdog degenerative changes of successive karyopyknosis, karyorrhexis and karyolysis. The nuclear envelope and the two layers of cell membranes separating the ingested cell from the phagocytic cell were

and The

destroyed, and finally, a phagocytic vacuole was formed within the phagocytic cell.

The molecular mechanisms underlying apoptosis are poorly understood at this time. However, there are several models of apoptotic initiation that are now accepted. Apoptosis has been found to be induced via the stimulation of several different cell surface receptors in association with caspase activation. For example, the CD95(APO-Ifas) receptor ligand system is a critical mediator of several physiological and pathophysiological processes, including homeostasis of the peripheral lymphoid compartment and CTL - mediated target cell killing . Upon cross-linking by ligand or agonist antibody, the fas receptor initiates a signal transduction cascade which leads to caspase-dependent programmed cell death. The simplest way to observe this phenomenon in vitro is to use a cell permeant DNA-staining fluorescent dye such as Hoechst 33342, which allows a striking visualization of the

research on the mechanisms of follicular apoptosis after the EMF exposure.

dying granulosa cells in the ovarian follicles is unclear.

chromatin condensation (Gartner and Hiatt 2001).

question. Based on ultrastructural criteria traditionally associated with apoptosis (Wyllie et al. 1972), oocyte death should be assigned to a different class of physiological cell death. Such variations in the mechanisms of cell death are becoming more widely accepted (Chernoff et al. 1992) and will be the subject of future investigations.

In the present study, we characterized the degeneration of rat ovarian tissue after the exposure to EMF, cell death of the interstitial tissue in ovary was shown to be apoptotic by morphological criteria with TEM. Taken together, our results suggest that apoptosis play a critical role in the degeneration of in situ ovarian cortical and interstitial tissue, after the exposure to EMF.

Some follicles with intact oocytes contain several layer of granulosa cells and fail to form follicles consisting of multiple layers of granulosa cells. We hypothesized that this lack of increase in granulosa cell number is due to either a lack of granulosa cell proliferation or to an increase in granulosa cell apoptosis. In contrast, ovary demonstrated large number of TUNEL-positive granulosa cells (Fig. 12), suggesting that most of the granulosa cells especially near the lumen are apoptotic cells. It appears that soon after oocyte degeneration, granulosa cell begins to undergo apoptosis in the follicles of the EMF-exposed ovary.

In summary, EMF-induced changes in ovary may interfere with oogenesis, fertility, and is an indication of the cytotoxic effect of EMF on maturation of oocytes.

In EMF-exposed rats, granulosa cells have a nucleus with condensed chromatin, apoptotic bodies and several autophagic vacuoles. The presence of granulosa cells with condensed nuclei and their separation from zona pellucida and neighbouring cells, corresponds with the characteristic of apoptotic cells and constitutes the classical land mark of follicular atresia (Hardwick 2004; Tilly et al. 1992; Hurwitz and Adashi 1991). While the proportion of granulosa cells with condensed nuclei was low in control group, this was evidenced by few number of apoptotic cells. This study demonstrates induction of programmed cell death by EMF and suggest a role for EMF in increasing of follicular atresia in rat ovary.

Based on the sequential ultrastructural observations of this study as well as previous works, the following model is proposed to explain the initiation of apoptosis in ovaries. Alterations occur in the granulosa cells include; nuclear condensation, apoptotic body formation and blebbing of the cytoplasm (present study and Peluso et al. 1977). As the follicle enters into the apoptotic changes, pyknotic nuclei, apoptotic bodies and numerous autophagic vacuoles develop. The autophagic vacuoles are associated with the granulosa cells close to the basal lamina. The fact that these vacuoles contain acid phosphatase activity indicates that they are lysosomal in origin (Elfont et al. 1977) and in part responsible for the deterioration of the granulosa cell layer. Autophagic vacuoles are also observed in the thecal layers, and interstitial tissue particulary associated with the thecal cells and interstitial cells near the blood vessels. The deterioration of both granulosa and thecal cell layers is also enhanced by invasion of macrophages which occurs after many of the granulosa cells have undergone apoptosis. From all morphological standpoints cytoplasmic bodies apparently corresponding to phagolysosomes, these large cells have been identified as macrophages. It has been proposed that such cells induce the invasion of macrophages (Byskov 1974) possibly by releasing a chemotactic factor (Gaytan et al. 1998). Regarding the mechanism whereby macrophages promote granulosa cell change it is postulated that macrophages have the capacity to produce oxidative products such as nitric oxide (Bredt et al. 1994), superoxide radicals, and hydrogen

question. Based on ultrastructural criteria traditionally associated with apoptosis (Wyllie et al. 1972), oocyte death should be assigned to a different class of physiological cell death. Such variations in the mechanisms of cell death are becoming more widely accepted

In the present study, we characterized the degeneration of rat ovarian tissue after the exposure to EMF, cell death of the interstitial tissue in ovary was shown to be apoptotic by morphological criteria with TEM. Taken together, our results suggest that apoptosis play a critical role in the degeneration of in situ ovarian cortical and interstitial tissue, after the

Some follicles with intact oocytes contain several layer of granulosa cells and fail to form follicles consisting of multiple layers of granulosa cells. We hypothesized that this lack of increase in granulosa cell number is due to either a lack of granulosa cell proliferation or to an increase in granulosa cell apoptosis. In contrast, ovary demonstrated large number of TUNEL-positive granulosa cells (Fig. 12), suggesting that most of the granulosa cells especially near the lumen are apoptotic cells. It appears that soon after oocyte degeneration,

In summary, EMF-induced changes in ovary may interfere with oogenesis, fertility, and is

In EMF-exposed rats, granulosa cells have a nucleus with condensed chromatin, apoptotic bodies and several autophagic vacuoles. The presence of granulosa cells with condensed nuclei and their separation from zona pellucida and neighbouring cells, corresponds with the characteristic of apoptotic cells and constitutes the classical land mark of follicular atresia (Hardwick 2004; Tilly et al. 1992; Hurwitz and Adashi 1991). While the proportion of granulosa cells with condensed nuclei was low in control group, this was evidenced by few number of apoptotic cells. This study demonstrates induction of programmed cell death by

Based on the sequential ultrastructural observations of this study as well as previous works, the following model is proposed to explain the initiation of apoptosis in ovaries. Alterations occur in the granulosa cells include; nuclear condensation, apoptotic body formation and blebbing of the cytoplasm (present study and Peluso et al. 1977). As the follicle enters into the apoptotic changes, pyknotic nuclei, apoptotic bodies and numerous autophagic vacuoles develop. The autophagic vacuoles are associated with the granulosa cells close to the basal lamina. The fact that these vacuoles contain acid phosphatase activity indicates that they are lysosomal in origin (Elfont et al. 1977) and in part responsible for the deterioration of the granulosa cell layer. Autophagic vacuoles are also observed in the thecal layers, and interstitial tissue particulary associated with the thecal cells and interstitial cells near the blood vessels. The deterioration of both granulosa and thecal cell layers is also enhanced by invasion of macrophages which occurs after many of the granulosa cells have undergone apoptosis. From all morphological standpoints cytoplasmic bodies apparently corresponding to phagolysosomes, these large cells have been identified as macrophages. It has been proposed that such cells induce the invasion of macrophages (Byskov 1974) possibly by releasing a chemotactic factor (Gaytan et al. 1998). Regarding the mechanism whereby macrophages promote granulosa cell change it is postulated that macrophages have the capacity to produce oxidative products such as nitric oxide (Bredt et al. 1994), superoxide radicals, and hydrogen

granulosa cell begins to undergo apoptosis in the follicles of the EMF-exposed ovary.

(Chernoff et al. 1992) and will be the subject of future investigations.

an indication of the cytotoxic effect of EMF on maturation of oocytes.

EMF and suggest a role for EMF in increasing of follicular atresia in rat ovary.

exposure to EMF.

peroxide (Sugino et al. 1996); and in addition, macrophage-derived cytokines such as transforming growth factor and have been found to induce apoptosis in ovarian cells (Foghi et al. 1996). The present study describe not only the ultrastracture of follicle in rat ovaries but also the increased degenerative changes within follicle committed to undergo apoptosis after EMFexposure (Roshangar and Soleimani Rad, 2007). In support of this idea several studies reported (Roshangar and Soleimani Rad 2001; Kaipia et al. 1997; Hughes and Gorospe 1991; Zamboni 1972) increased of macrophages after the EMF exposure. It is believed that the cell fragments produced by apoptosis are phagocytosed by macrophages. These macrophages do not release cytokines that would initiate an inflammatory response (Andrew et al. 1998).Thus the granulosa and thecal cells would be destroyed, leaving only fibroblasts and other connective tissue elements to represent the follicular wall, thereby transforming the follicle into a cystic follicle. It has been reported that macrophages were involved in the apoptotic process (Leonardo and Skeel 1980). This clearly indicates that increased number of macrophages in experimental group (Soleimani Rad and Roshangar 2000) potentially increases cell damages. Using rat ovaries, the present study confirms that the follicular granulosa cells undergo apoptosis after the EMF exposure on the basis of their microscopic features, i.e., condensation of nuclear chromatin to the margin of the nucleus, and presence of apoptotic bodies, both changes being characteristic of apoptosis (Ker et al. 1972), even more evidence is occurring in parallel with the above- mentioned morphological changes. As far as we aware, the fate of the dying granulosa cells in the ovarian follicles is unclear.

In the present study in rats, we demonstrated that large amount apoptotic and internalized granulosa cells and their fragments. The factors responsible for this basic difference are as yet unknown. In summary, this model is proposed to foster and more clearly focus future research on the mechanisms of follicular apoptosis after the EMF exposure.

Our results demonstrate that a remarkable proportion of oocytes in the rat ovary degenerate during the EMF exposure by the mechanism of apoptosis. This is already evident at experimental period, with a high number of apoptotic oocytes and increasing of macrophages and autophagic vacuoles in some occasional granulosa cells, and several lipid droplets in thecal and luteal cells. Previous TEM studies (Takeo and Hokano 1995) suggested that the process of apoptosis of the ingested cells was assumed to progress through the following steps. The nuclei of ingested cells underdog degenerative changes of successive karyopyknosis, karyorrhexis and karyolysis. The nuclear envelope and the two layers of cell membranes separating the ingested cell from the phagocytic cell were destroyed, and finally, a phagocytic vacuole was formed within the phagocytic cell.

The molecular mechanisms underlying apoptosis are poorly understood at this time. However, there are several models of apoptotic initiation that are now accepted. Apoptosis has been found to be induced via the stimulation of several different cell surface receptors in association with caspase activation. For example, the CD95(APO-Ifas) receptor ligand system is a critical mediator of several physiological and pathophysiological processes, including homeostasis of the peripheral lymphoid compartment and CTL - mediated target cell killing . Upon cross-linking by ligand or agonist antibody, the fas receptor initiates a signal transduction cascade which leads to caspase-dependent programmed cell death. The simplest way to observe this phenomenon in vitro is to use a cell permeant DNA-staining fluorescent dye such as Hoechst 33342, which allows a striking visualization of the chromatin condensation (Gartner and Hiatt 2001).

Environmental Electromagnetic Field and Female Fertility 81

functionally important substrates are not yet clearly defined. Persuasive evidence that these proteases are involved in most examples of apoptotic cell death has come from the ability of specific caspase inhibitors to block cell death, as well as the demonstration that knockout mice lacking caspase 3, 8 and 9 fail to complete normal embryonic development. A critical issue is how caspases become initially activated, which seems to be an irreversible commitment towards death. It seems that aggregation of some pro-caspases (those with large pro- domains) allows them to become autoactivated. Recent experiments make it clear that mitochondria are involved in one major pathway involving activation of pro- caspase-9. Other experiments show that ligands crosslinking death receptors such as Fas trigger formation of a cytoplasmic complex in which pro-caspase-8 is aggregated and activated. In both cases these initiator caspases in turn activate a cascade of other pro-caspases leading to

While there is much to be learned about the molecular pathways leading to apoptotic cell death, it is increasingly clear that cell death is a normal part of normal biological processes. This had not been appreciated until relatively recently, and our understanding of such death, and our ability to manipulate it, could allow therapeutic intervention in major diseases such as

We can only speculate how our EMF would fit into one of these models for induction of apoptosis. One such model is apoptotic initiation by intracellular perturbation. Examples of this model are ionizing radiation and chemotherapeutic ionizing agents, which cause DNA damage and initiation of apoptosis. An electromagnetic field could have a similar effect

Additionally, it has been shown that the overexpression of c-myc portion leads to apoptosis. It is interesting that EMFs have been shown to increase specifically transcription of c-myc in several cell lines (Lin et al 1998). This is just one of the numerous possible mechanisms that could be inducing apoptosis. Another possibility is EMF-inducing apoptosis is mediated through the production of free radicals. It is shown that EMF exposure may result in production of free radicals (Lucia et al. 2004; Brocklehurst and McLauchlan 1996; Grundler et al. 1992; Alexander 1954). It is also shown that addition of antioxidants, such as vitamine E reduces EMF-induced changes *in vivo* and *in vitro* (Mohammadnejad and Soleimani Rad 2010). On the other hand free radicals as an inducer of apoptosis is also established (Formica

The findings with TEM about apoptosis-induced by EMF is confirmed using TUNEL assay. TUNEL positive cells are localized in granulosa layer, thecal cells, luteal cells and interstitial cells. The localization of apoptotic cells are well correlate with TEM studies. It is proposed that combining other methods such as microscopic evaluation of morphological changes

Although the mechanisms underlying follicular atresia are not well known at this time, DNA damage, which can be initiated by oxidative free radicals, has been proposed as a possible mechanism that leads to the activation of the apoptotic cascade in atretic follicles (Gougeon 1996). Macrophages have the capacity to produce oxidative products such as nitric oxide (Bredt and Synder 1994), superoxide radicals, and hydrogen peroxide (Sugino et al. 1996). Macrophage-derived cytokines such as transforming growth factor α induce

with TUNEL POD test can substantiate the specificity of results.

apoptosis in ovarian cells (Foghi et al. 1997).

cancer, heart disease, stroke, AIDS, autoimmunity, degenerative diseases, and others.

death (Andrew et al. 1998).

(Norman et al. 1997).

and Silvestri 2004).

 Apoptosis is over 20 times faster than mitosis. Seeing of dying cells in vivo are therefore rare. Apoptotic cells are engulfed and degraded by neighboring cells without a trace. For cell homeostasis to be maintained, a balance between the increase (by differentiation from precursors and by proliferation) and decrease (by further differentiation and cell death) in the number of a cell population has to be neatly balanced. If mitosis proceed without cell death, an 80-year-old person would have 2 tons of bone marrow and lymph nodes, and a gut 16 Km long.

Apoptotic death can be triggered by a wide variety of stimuli, and not all cells necessarily will die in response to the same stimulus. Among the more studied death stimuli is DNA damage (by irradiation or drugs used for cancer chemotherapy), which in many cells leads to apoptotic death via a pathway dependent on p53. Some hormones such as corticosteroids lead to death in particular cells (e.g., thymocytes), although other cell types may be stimulated. Some cell types express Fas, a surface protein which initiates an intracellular death signal in response to cross-linking. In other cases cells appear to have a default death pathway which must be actively blocked by a survival factor in order to allow cell survival, a survival factor normally binds to its cell surface receptor. When the survival factor is removed, the default apoptotic death program is triggered (Andrew et al. 1998).

Biochemical correlates of these morphological features have emerged during the subsequent years of study of this phenomenon. The first and most dramatic is DNA fragmentation, which was described by Brocklehurst 1996; McLauchlan 1981; and Wyllie 1980. When DNA from apoptotically dying cells was subjected to agarose gel electrophoresis, ladders with – 200 bp repeats were observed, corresponding histone protection in the nucleosomes of native chromatin. Subsequent pulsed field gel techniques have revealed earlier DNA cleavage patterns into larger fragments. Since even a few double stranded DNA breaks will render the cell unable to undergo mitosis successfully, such DNA fragmentation can be regarded as a biochemical definition of death. However, in some apoptotic systems (e.g., Fas killing of tumor cells) artificially enucleated cells lacking a nucleus still die, showing that the nucleus is not always necessary for apoptotic cell death.

The changes in the apoptotic cell which trigger phagocytosis by non-activated macrophages have been investigated by several groups. Macrophages appear to recognize apoptotic cells via several different recognition systems, which seem to recognize recognition used preferentially by different macrophage subpopulations. There is good evidence that apoptotic cells lose the normal phospholipid asymmetry in their plasma membrane, as manifested by the exposure of normally inward-facing phosphatidyl serine on the external face of the bilayer. Macrophages can recognize this exposed lipid head group via an unknown receptor, triggering phagocytosis.

Another biochemical landmark of apoptotic death which increasingly appears general is the activation of caspases, which are cysteine proteases related to *ced-3,* the "death gene" of the nematode *Caenorhabditis elegans.* caspases seem to be widely expressed in an inactive proenzyme form in most cells. Their proteolytic activity is characterized by their unusual ability to cleave proteins at aspartic acid residues, although different caspases have different fine specificities involving recognition of neighboring amino acids. Active caspases can often activate other pro-caspases, allowing initiation of a protease cascade. While several protein substrates have been shown to be cleaved by caspases during apoptotic death, the

 Apoptosis is over 20 times faster than mitosis. Seeing of dying cells in vivo are therefore rare. Apoptotic cells are engulfed and degraded by neighboring cells without a trace. For cell homeostasis to be maintained, a balance between the increase (by differentiation from precursors and by proliferation) and decrease (by further differentiation and cell death) in the number of a cell population has to be neatly balanced. If mitosis proceed without cell death, an 80-year-old person would have 2 tons of bone marrow and lymph nodes, and a

Apoptotic death can be triggered by a wide variety of stimuli, and not all cells necessarily will die in response to the same stimulus. Among the more studied death stimuli is DNA damage (by irradiation or drugs used for cancer chemotherapy), which in many cells leads to apoptotic death via a pathway dependent on p53. Some hormones such as corticosteroids lead to death in particular cells (e.g., thymocytes), although other cell types may be stimulated. Some cell types express Fas, a surface protein which initiates an intracellular death signal in response to cross-linking. In other cases cells appear to have a default death pathway which must be actively blocked by a survival factor in order to allow cell survival, a survival factor normally binds to its cell surface receptor. When the survival factor is

Biochemical correlates of these morphological features have emerged during the subsequent years of study of this phenomenon. The first and most dramatic is DNA fragmentation, which was described by Brocklehurst 1996; McLauchlan 1981; and Wyllie 1980. When DNA from apoptotically dying cells was subjected to agarose gel electrophoresis, ladders with – 200 bp repeats were observed, corresponding histone protection in the nucleosomes of native chromatin. Subsequent pulsed field gel techniques have revealed earlier DNA cleavage patterns into larger fragments. Since even a few double stranded DNA breaks will render the cell unable to undergo mitosis successfully, such DNA fragmentation can be regarded as a biochemical definition of death. However, in some apoptotic systems (e.g., Fas killing of tumor cells) artificially enucleated cells lacking a nucleus still die, showing that the

The changes in the apoptotic cell which trigger phagocytosis by non-activated macrophages have been investigated by several groups. Macrophages appear to recognize apoptotic cells via several different recognition systems, which seem to recognize recognition used preferentially by different macrophage subpopulations. There is good evidence that apoptotic cells lose the normal phospholipid asymmetry in their plasma membrane, as manifested by the exposure of normally inward-facing phosphatidyl serine on the external face of the bilayer. Macrophages can recognize this exposed lipid head group via an

Another biochemical landmark of apoptotic death which increasingly appears general is the activation of caspases, which are cysteine proteases related to *ced-3,* the "death gene" of the nematode *Caenorhabditis elegans.* caspases seem to be widely expressed in an inactive proenzyme form in most cells. Their proteolytic activity is characterized by their unusual ability to cleave proteins at aspartic acid residues, although different caspases have different fine specificities involving recognition of neighboring amino acids. Active caspases can often activate other pro-caspases, allowing initiation of a protease cascade. While several protein substrates have been shown to be cleaved by caspases during apoptotic death, the

different

removed, the default apoptotic death program is triggered (Andrew et al. 1998).

nucleus is not always necessary for apoptotic cell death.

unknown receptor, triggering phagocytosis.

gut 16 Km long.

functionally important substrates are not yet clearly defined. Persuasive evidence that these proteases are involved in most examples of apoptotic cell death has come from the ability of specific caspase inhibitors to block cell death, as well as the demonstration that knockout mice lacking caspase 3, 8 and 9 fail to complete normal embryonic development. A critical issue is how caspases become initially activated, which seems to be an irreversible commitment towards death. It seems that aggregation of some pro-caspases (those with large pro- domains) allows them to become autoactivated. Recent experiments make it clear that mitochondria are involved in one major pathway involving activation of pro- caspase-9. Other experiments show that ligands crosslinking death receptors such as Fas trigger formation of a cytoplasmic complex in which pro-caspase-8 is aggregated and activated. In both cases these initiator caspases in turn activate a cascade of other pro-caspases leading to death (Andrew et al. 1998).

While there is much to be learned about the molecular pathways leading to apoptotic cell death, it is increasingly clear that cell death is a normal part of normal biological processes. This had not been appreciated until relatively recently, and our understanding of such death, and our ability to manipulate it, could allow therapeutic intervention in major diseases such as cancer, heart disease, stroke, AIDS, autoimmunity, degenerative diseases, and others.

We can only speculate how our EMF would fit into one of these models for induction of apoptosis. One such model is apoptotic initiation by intracellular perturbation. Examples of this model are ionizing radiation and chemotherapeutic ionizing agents, which cause DNA damage and initiation of apoptosis. An electromagnetic field could have a similar effect (Norman et al. 1997).

Additionally, it has been shown that the overexpression of c-myc portion leads to apoptosis. It is interesting that EMFs have been shown to increase specifically transcription of c-myc in several cell lines (Lin et al 1998). This is just one of the numerous possible mechanisms that could be inducing apoptosis. Another possibility is EMF-inducing apoptosis is mediated through the production of free radicals. It is shown that EMF exposure may result in production of free radicals (Lucia et al. 2004; Brocklehurst and McLauchlan 1996; Grundler et al. 1992; Alexander 1954). It is also shown that addition of antioxidants, such as vitamine E reduces EMF-induced changes *in vivo* and *in vitro* (Mohammadnejad and Soleimani Rad 2010). On the other hand free radicals as an inducer of apoptosis is also established (Formica and Silvestri 2004).

The findings with TEM about apoptosis-induced by EMF is confirmed using TUNEL assay. TUNEL positive cells are localized in granulosa layer, thecal cells, luteal cells and interstitial cells. The localization of apoptotic cells are well correlate with TEM studies. It is proposed that combining other methods such as microscopic evaluation of morphological changes with TUNEL POD test can substantiate the specificity of results.

Although the mechanisms underlying follicular atresia are not well known at this time, DNA damage, which can be initiated by oxidative free radicals, has been proposed as a possible mechanism that leads to the activation of the apoptotic cascade in atretic follicles (Gougeon 1996). Macrophages have the capacity to produce oxidative products such as nitric oxide (Bredt and Synder 1994), superoxide radicals, and hydrogen peroxide (Sugino et al. 1996). Macrophage-derived cytokines such as transforming growth factor α induce apoptosis in ovarian cells (Foghi et al. 1997).

Environmental Electromagnetic Field and Female Fertility 83

For biological effects of EMF, non thermal mechanisms are also proposed. These include: 1) Free radical formation. 2) Removal of Ca ions from membranes and and making them more likely to tear and keak. 3) DNAase leaking through the lysosomal membrane and DNA damage. 4) Leakage of Ca ions into the cytosol and acting as metabolic stimulant (Goldsworthy 2007). With regard to the effect of' free radicals, it has been proposed that free radical could affect membrane integrity, produce DNA damage, and protein structures (Alexander and Charlesby 1954). This type of changes, would result in cell damage and

No conclusion can be drawn for electromagnetic fields and radiofrequencies because of lack of data, but there is no convincing evidence today that EMFs of the sort pregnant women or potential fathers meet in occupational or daily life exposures do any harm to the human

Additionally, it has been hypothesized that electromagnetic fields initially affect cells at their surface, since these low energy fields cannot directly access the cell interior because of the high resistance of the cell plasma membrane (Luben et al. 1982). The proteins that span the width of the plasma membrane therefore, have been hypothesized to act as potential sensors of ELF electromagnetic so that their actions may be transmitted to intracellular

Moreover, transformed cells and normal ones show different electrical characteristics as extensively documented by several scientists (Capko et al. 1996; Shulyakovskaya et al. 1993; Goller et al. 1986). These results led to the hypothesis that EMF of more than 1 mT, may through their effect on motion of charged matter, have a selective action on cell signaling which influences cell survival mechanisms in transformed cell, inhibiting their growth and differentiation (Tofani 1999). Also EMF have been shown to affect different aspects of biomolecular synthesis in cell, including the kinetics of DNA, RNA, and protein production (Libof 1985). Increased DNA and proteoglycan synthesis have been observed in chondrocytes (Rodan et al. 1978). In fibroblasts, low – intensity electric and magnetic fields altered collagen and proteoglycan synthesis (Farndale and Murray 1985). A complex range of effects was observed depending on the exact magnetic field

Gap junctions are specialized areas of the plasma membranes between two contiguous cells where a" pore" is formed that allows for the passage of small molecules between cells (Dean et al. 2002; Loewenstein 1979). These gap junctions are composed of proteins called connexins which have extracellular regions that attach to other connexin proteins of a contiguous cell as well as having intramembranous and cytoplasmic domains. Thus connexin proteins could also be targets of electromagnetic fields. Gap junctions have been ultrastructurally described in bone (Doty 1981). They occur among osteoblasts and osteocytes (Takahashi 2002; Boone and Tsang 1997). It has been noted that gap junctions can be regulated by change in cellular Ca2+ concentration. Micromolar concentration of Ca2+ have been demonstrated to decrease gap junction intercellular communication in a variety of tissues including cardiac muscle and liver (Li et al. 1999; Hertzberg et al. 1981). Thus it would be important to understand if changes in intracellular Ca2+ metabolism that may occur with exposure to ELF magnetic fields would be related to alterations in gap junction

induction of apoptosis (Formica and Silvestri 2004).

dependent intercellular communication (Luben 1991).

reproductive process (Cheronff et al. 1992).

enzymes and organelles (Adey 1990).

configurations.

In addition to the ovarian follicles the apoptotic status and nuclear condensation is observed in corpora lutea and ovarian stroma. These findings was also confirmed with the presence of TUNEL positive cells. Ultrastractural characteristics of apoptotic cells in the corpora lutea in this study is similar to those reported by Shikone et. al. 1996. Another finding in the present study was the presence of numerous macrophages in the stroma and granulosa layer in EMF exposed rats. Since the presence of macrophages in the granulosa layer of atretic follicles and the degenerating corpora lutea of rats was demonstrated by Bulmer (Bulmer 1964), macrophages have been thought to scavenge degenerated cells in the ovary (Lauber et al 2004; Anderson et al. 2003).

In addition to apoptotic changes induced by EMF exposure the other ultrastractural changes include mitochondrial disruption, condensation and or their cristae disappearance, rER dilatation and in some occasion, cytoplasmic membrane dissolution. All these alterations are the sign of cell damage and necrosis rather than apoptosis. The rational explanation would be that: EMF induces both apoptosis and necrosis depending on its strength and cell types. For example typical degenerative changes were never observed in oocytes, while it occurred in many granulosa cells. The cytotoxic effect of EMF could be attributed to its production of local heat and free radicals. This hypothesis is evidenced by the studies have shown that EMF could produce heat and free radicals (Dandrea et al. 2003; Grundler et al. 1992; Alexander and Charlesby 1954).

In this regards, it has been shown that electromagnetic fields from power lines, household currents and video display terminals, microwaves, and ultrasound have also been studied with regard to their reproductive risks. Biological plausibility plays an even more important role in their evaluation than with ionizing radiation. EMF has the capacity to produce hyperthermia, which is a proven reproductive toxin. Numerous animal experiments have demonstrated that intrauterine exposures to hypertheimia from microwaves and ultrasound and EMFs can produce malformations, growth retardation, and embryonic loss. But the usual population exposures to EMFs are below the exposures that result in hyperthermia. Furthermore, those mechanisms that are involved in reproductive toxicity, such as cytotoxicity and abnormal differentiation and cell migration do not occur at the population exposures to these agents. Evaluations of pregnancy loss from intrauterine exposures to environmental toxicants presents special problems, especially if it is the only reproductive effect being evaluated. Many studies have ignored the basic concepts of reproductive toxicology and the biological plausibility of their findings. Investigators should be cautious about biological Investigator epidemiological studies dealing with pregnancy loss without concurrent collecting other reproductive endpoints. Studies evaluating multiple reproductive endpoints have markers to assist them in determining the validity of the fertility loss data (Byene 1999).

In summary, the present data suggest that the currently applied EMF levels under certain circumstances might induce biological effects. Results indicate that the genetic constitution of cells determined by loss of P53 function can influence EMF related cellular responses. Whereas wild-type cells were insensitive. It remains to be elucidated, whether EMF induced changes of expression levels of regulatory genes may be compensated or normalized, or would result in sustained biological effects in vivo. Further studies are needed to analyze the whole transcription of EMF exposed cells by genomics technologies, such as cDNA microchips or serial analysis of gene expression (SAGE), because of conflicting epidemiological data on human EMF exposure (Repacholi 1998).

In addition to the ovarian follicles the apoptotic status and nuclear condensation is observed in corpora lutea and ovarian stroma. These findings was also confirmed with the presence of TUNEL positive cells. Ultrastractural characteristics of apoptotic cells in the corpora lutea in this study is similar to those reported by Shikone et. al. 1996. Another finding in the present study was the presence of numerous macrophages in the stroma and granulosa layer in EMF exposed rats. Since the presence of macrophages in the granulosa layer of atretic follicles and the degenerating corpora lutea of rats was demonstrated by Bulmer (Bulmer 1964), macrophages have been thought to scavenge degenerated cells in the ovary (Lauber et

In addition to apoptotic changes induced by EMF exposure the other ultrastractural changes include mitochondrial disruption, condensation and or their cristae disappearance, rER dilatation and in some occasion, cytoplasmic membrane dissolution. All these alterations are the sign of cell damage and necrosis rather than apoptosis. The rational explanation would be that: EMF induces both apoptosis and necrosis depending on its strength and cell types. For example typical degenerative changes were never observed in oocytes, while it occurred in many granulosa cells. The cytotoxic effect of EMF could be attributed to its production of local heat and free radicals. This hypothesis is evidenced by the studies have shown that EMF could produce heat and free radicals (Dandrea et al. 2003; Grundler et al. 1992;

In this regards, it has been shown that electromagnetic fields from power lines, household currents and video display terminals, microwaves, and ultrasound have also been studied with regard to their reproductive risks. Biological plausibility plays an even more important role in their evaluation than with ionizing radiation. EMF has the capacity to produce hyperthermia, which is a proven reproductive toxin. Numerous animal experiments have demonstrated that intrauterine exposures to hypertheimia from microwaves and ultrasound and EMFs can produce malformations, growth retardation, and embryonic loss. But the usual population exposures to EMFs are below the exposures that result in hyperthermia. Furthermore, those mechanisms that are involved in reproductive toxicity, such as cytotoxicity and abnormal differentiation and cell migration do not occur at the population exposures to these agents. Evaluations of pregnancy loss from intrauterine exposures to environmental toxicants presents special problems, especially if it is the only reproductive effect being evaluated. Many studies have ignored the basic concepts of reproductive toxicology and the biological plausibility of their findings. Investigators should be cautious about biological Investigator epidemiological studies dealing with pregnancy loss without concurrent collecting other reproductive endpoints. Studies evaluating multiple reproductive endpoints have markers to assist them in determining the validity of the fertility loss data (Byene 1999). In summary, the present data suggest that the currently applied EMF levels under certain circumstances might induce biological effects. Results indicate that the genetic constitution of cells determined by loss of P53 function can influence EMF related cellular responses. Whereas wild-type cells were insensitive. It remains to be elucidated, whether EMF induced changes of expression levels of regulatory genes may be compensated or normalized, or would result in sustained biological effects in vivo. Further studies are needed to analyze the whole transcription of EMF exposed cells by genomics technologies, such as cDNA microchips or serial analysis of gene expression (SAGE), because of conflicting

epidemiological data on human EMF exposure (Repacholi 1998).

al 2004; Anderson et al. 2003).

Alexander and Charlesby 1954).

For biological effects of EMF, non thermal mechanisms are also proposed. These include: 1) Free radical formation. 2) Removal of Ca ions from membranes and and making them more likely to tear and keak. 3) DNAase leaking through the lysosomal membrane and DNA damage. 4) Leakage of Ca ions into the cytosol and acting as metabolic stimulant (Goldsworthy 2007). With regard to the effect of' free radicals, it has been proposed that free radical could affect membrane integrity, produce DNA damage, and protein structures (Alexander and Charlesby 1954). This type of changes, would result in cell damage and induction of apoptosis (Formica and Silvestri 2004).

No conclusion can be drawn for electromagnetic fields and radiofrequencies because of lack of data, but there is no convincing evidence today that EMFs of the sort pregnant women or potential fathers meet in occupational or daily life exposures do any harm to the human reproductive process (Cheronff et al. 1992).

Additionally, it has been hypothesized that electromagnetic fields initially affect cells at their surface, since these low energy fields cannot directly access the cell interior because of the high resistance of the cell plasma membrane (Luben et al. 1982). The proteins that span the width of the plasma membrane therefore, have been hypothesized to act as potential sensors of ELF electromagnetic so that their actions may be transmitted to intracellular enzymes and organelles (Adey 1990).

Moreover, transformed cells and normal ones show different electrical characteristics as extensively documented by several scientists (Capko et al. 1996; Shulyakovskaya et al. 1993; Goller et al. 1986). These results led to the hypothesis that EMF of more than 1 mT, may through their effect on motion of charged matter, have a selective action on cell signaling which influences cell survival mechanisms in transformed cell, inhibiting their growth and differentiation (Tofani 1999). Also EMF have been shown to affect different aspects of biomolecular synthesis in cell, including the kinetics of DNA, RNA, and protein production (Libof 1985). Increased DNA and proteoglycan synthesis have been observed in chondrocytes (Rodan et al. 1978). In fibroblasts, low – intensity electric and magnetic fields altered collagen and proteoglycan synthesis (Farndale and Murray 1985). A complex range of effects was observed depending on the exact magnetic field configurations.

Gap junctions are specialized areas of the plasma membranes between two contiguous cells where a" pore" is formed that allows for the passage of small molecules between cells (Dean et al. 2002; Loewenstein 1979). These gap junctions are composed of proteins called connexins which have extracellular regions that attach to other connexin proteins of a contiguous cell as well as having intramembranous and cytoplasmic domains. Thus connexin proteins could also be targets of electromagnetic fields. Gap junctions have been ultrastructurally described in bone (Doty 1981). They occur among osteoblasts and osteocytes (Takahashi 2002; Boone and Tsang 1997). It has been noted that gap junctions can be regulated by change in cellular Ca2+ concentration. Micromolar concentration of Ca2+ have been demonstrated to decrease gap junction intercellular communication in a variety of tissues including cardiac muscle and liver (Li et al. 1999; Hertzberg et al. 1981). Thus it would be important to understand if changes in intracellular Ca2+ metabolism that may occur with exposure to ELF magnetic fields would be related to alterations in gap junction dependent intercellular communication (Luben 1991). ELF

Environmental Electromagnetic Field and Female Fertility 85

There is no doubt that cell damages are the basis of all disorders occurs after EMF exposure. In support of our findings Sandra et. al. (Sandra et al. 2000) suggested that EMF-exposure might impair mammalian female reproductive potentiality by reducing the capacity of the follicles to reach a developmental stage that is an essential pre requisite for reproductive

The acceleration of cell damage with EMF-exposure in reproductive organs in rathas previously been reported (Roshangar and Soleimani Rad 2002; Soleimani Rad and Roshangar 2000; Byskov 1974). To elucidate the mechanisms underlying the acceleration of cell damage (Armstrong et al. 2001; Chun et al. 1996), in the present study, the earlier step of cell damage on the cellular membrane integrity, mitochondrial features, appearance of apoptotic bodies, nucleus condensation, and lipid droplet accumulation is investigated with EM and TUNEL assay. Each of these end points showed a parallel correlation with

These findings may explain the acceleration and increasing of apoptotic process by the conditioning dose. Contrary to the acceleration of apoptosis shown in the present study, in some types of cells, such as malignant cells (Ohnishi et al. 2002) and mouse spleen cells (Takahashi et al. 2002). As a possible mechanism, the attenuation of P53 response has been postulated (Dean et al. 2002; Doty 1981). In cellular responses to ionizing radiation, P53 plays very important roles. It regulates DNA repair, which leads cells to die. The partial involvement of P53 in the regulation of apoptosis is also suggested (Perez et al. 1999;

The recent evidence pointing to the role of caspases in activating DNA degradation (Green and Reed 1998; Liu et al 1997) suggest that in order for ovarian cells to complete the apoptotic program they must contain caspase-3, and an endogenous nuclear DNAase. Boone et.al. (Boone and Yan 1995) demonstrated that granulosa and luteal cells contain endogenous nuclear DNAase, and they hypothesized that these cells would therefore only require a signal to activate this enzyme in order to degrade their DNA in an apoptotic

In conclusion, we have shown that EMF-exposure causes a large proportion of oocytes in the rat ovary to degenerate by a mechanism similar to apoptosis. This is evident in the EMFexposed group with a large number of degenerative oocytes. Other findings of the study are: an increased number of macrophages; autophagic vacuoles in some granulosa cells; and appearance of several lipid droplets in thecal and luteal cells. The present study has also shown the increased number of macrophages not only in the corpora lutea but also in the growing follicles in the EMF-exposed group. Based on our TEM, we have proposed a model

The aim of this work was to monitor the reproductive effect of exposure to a magnetic field in rat. Taken together, our results suggest that apoptosis plays a critical role in the degeneration of ovarian cortical tissue, luminal epithelium, glandular epithelium and stromal cells in uterus and luminal epithelium in fallopian tube. The present EMF- exposure model can be used when striving to find ways to improve the viability of ovarian tissue in

that can explain the initiation of apoptosis by EMF-exposure in ovaries.

apoptosis when the animals exposed to EMF for long time.

fashion (Laun et al. 2000; Boone and Tsang 1997).

success.

Vousden 2005).

**4.4 Conclusion** 

Research on the effects of electromagnetic fields on cells has known to alter some important physiological pathways. Of note are ionic conductances with such species as calcium ion (Ca2+) which is a known interacellular messenger in cell functions such as proliferation and intercellular communication. Specifically, with respect to extremely low-frequency (EMF) (<300 Hz) magnetic fields, Ca2+, uptake into lymphocytes was shown to increase (Lednev 1991). Walleczek (1992), summarized the effect of ELF magnetic fields, showing that such fields could either increase or decrease Ca2+ uptake into lymphocytes, depending on the time of exposure, the frequency and shape of the signal, and concomitant induced electric field intensity.

Mechanisms of interaction of ELF fields have been reviewed by Blank (1995), NRC (1996), Tenforde et al. (1996), and Valberg et al. (1997). A well-known mechanism of interaction of exposure of biological tissues to ELF fields is the induction of time- varying electric currents and fields. At sufficiently high levels, these can produce direct stimulation of excitable cells such as nerve and muscle cells. At the cellular level, the interaction induces voltages across the membranes of cells sufficient to stimulate nerves to conduct or muscles to contract. This mechanism accounts for the ability of humans and animals to perceive electric currents in their bodies and to experience electric shocks.

Our results have also been demonstrated the junctional changes that occur after EMFexposure (Figs. 7, 9). Moreover, it is postulated that developmental exposure to EMF may reduce oocyte differentiation and diminish folliculogenesis at earlier stages of oocyte and follicular nest formation. Both of which could result in decreasing of ovarian reservoir and thus the individual will be prone to subfertility in adulthood.

#### **4.3 EMF-Induced ultrastractural changes and apoptosis in uterus and fallopian tubes**

Other findings in the present study are the effects of EMF on the lining epithelium of endometrium, uterine glands and Fallopian tubes. Ultrastractural results from EMF exposed rats revealed that the height of epithelial and glandular cells both in uterus and Fallopian tubes were reduced, indicating metabolic activity of cells were decreased. On the other hand, there were condensation and cilliary loss, which could be considered as pre apoptotic changes.

This postulation was confirmed by apoptosis assay, using TUNEL reaction technique which was revealed apoptotic cells (TUNEL positive cells) in both endometrial surface and glandular epithelium of uterus and covering epithelium of Fallopian tubes. Ultrastractural changes corresponding to cytotoxic effect of EMF were also observed. These changes include; accumulation of numerous fat droplets in the secretory cells, presence of secondary lysosomes and morphological changes of mitochondria.

As we know, endometrial surface epithelium plays a key role in blastocyst implantation and an implantation window is required for the process of implantation to begin (Marti et al. 2001; Otasuki 2001). Additionally, increasing of secretion is usually occurs in preimplantation stage (Marti et al. 2001). Any changes in the amount and or nature of secretory substance would obviously affect implantation process. Similarly, the activity of ciliated and nonciliated cells in the covering of Fallopian tubes are very important factor for transport and early development of preimplant embryo. The structural changes produced by EMF could affect both development and transport of early embryo.

Research on the effects of electromagnetic fields on cells has known to alter some important physiological pathways. Of note are ionic conductances with such species as calcium ion (Ca2+) which is a known interacellular messenger in cell functions such as proliferation and intercellular communication. Specifically, with respect to extremely low-frequency (EMF) (<300 Hz) magnetic fields, Ca2+, uptake into lymphocytes was shown to increase (Lednev 1991). Walleczek (1992), summarized the effect of ELF magnetic fields, showing that such fields could either increase or decrease Ca2+ uptake into lymphocytes, depending on the time of exposure, the frequency and shape of the signal, and concomitant induced electric

Mechanisms of interaction of ELF fields have been reviewed by Blank (1995), NRC (1996), Tenforde et al. (1996), and Valberg et al. (1997). A well-known mechanism of interaction of exposure of biological tissues to ELF fields is the induction of time- varying electric currents and fields. At sufficiently high levels, these can produce direct stimulation of excitable cells such as nerve and muscle cells. At the cellular level, the interaction induces voltages across the membranes of cells sufficient to stimulate nerves to conduct or muscles to contract. This mechanism accounts for the ability of humans and animals to perceive electric currents in

Our results have also been demonstrated the junctional changes that occur after EMFexposure (Figs. 7, 9). Moreover, it is postulated that developmental exposure to EMF may reduce oocyte differentiation and diminish folliculogenesis at earlier stages of oocyte and follicular nest formation. Both of which could result in decreasing of ovarian reservoir and

**4.3 EMF-Induced ultrastractural changes and apoptosis in uterus and fallopian tubes**  Other findings in the present study are the effects of EMF on the lining epithelium of endometrium, uterine glands and Fallopian tubes. Ultrastractural results from EMF exposed rats revealed that the height of epithelial and glandular cells both in uterus and Fallopian tubes were reduced, indicating metabolic activity of cells were decreased. On the other hand, there were condensation and cilliary loss, which could be considered as pre apoptotic

This postulation was confirmed by apoptosis assay, using TUNEL reaction technique which was revealed apoptotic cells (TUNEL positive cells) in both endometrial surface and glandular epithelium of uterus and covering epithelium of Fallopian tubes. Ultrastractural changes corresponding to cytotoxic effect of EMF were also observed. These changes include; accumulation of numerous fat droplets in the secretory cells, presence of secondary

As we know, endometrial surface epithelium plays a key role in blastocyst implantation and an implantation window is required for the process of implantation to begin (Marti et al. 2001; Otasuki 2001). Additionally, increasing of secretion is usually occurs in preimplantation stage (Marti et al. 2001). Any changes in the amount and or nature of secretory substance would obviously affect implantation process. Similarly, the activity of ciliated and nonciliated cells in the covering of Fallopian tubes are very important factor for transport and early development of preimplant embryo. The structural changes produced

field intensity.

changes.

their bodies and to experience electric shocks.

thus the individual will be prone to subfertility in adulthood.

lysosomes and morphological changes of mitochondria.

by EMF could affect both development and transport of early embryo.

There is no doubt that cell damages are the basis of all disorders occurs after EMF exposure. In support of our findings Sandra et. al. (Sandra et al. 2000) suggested that EMF-exposure might impair mammalian female reproductive potentiality by reducing the capacity of the follicles to reach a developmental stage that is an essential pre requisite for reproductive success.

The acceleration of cell damage with EMF-exposure in reproductive organs in rathas previously been reported (Roshangar and Soleimani Rad 2002; Soleimani Rad and Roshangar 2000; Byskov 1974). To elucidate the mechanisms underlying the acceleration of cell damage (Armstrong et al. 2001; Chun et al. 1996), in the present study, the earlier step of cell damage on the cellular membrane integrity, mitochondrial features, appearance of apoptotic bodies, nucleus condensation, and lipid droplet accumulation is investigated with EM and TUNEL assay. Each of these end points showed a parallel correlation with apoptosis when the animals exposed to EMF for long time.

These findings may explain the acceleration and increasing of apoptotic process by the conditioning dose. Contrary to the acceleration of apoptosis shown in the present study, in some types of cells, such as malignant cells (Ohnishi et al. 2002) and mouse spleen cells (Takahashi et al. 2002). As a possible mechanism, the attenuation of P53 response has been postulated (Dean et al. 2002; Doty 1981). In cellular responses to ionizing radiation, P53 plays very important roles. It regulates DNA repair, which leads cells to die. The partial involvement of P53 in the regulation of apoptosis is also suggested (Perez et al. 1999; Vousden 2005). DNA

The recent evidence pointing to the role of caspases in activating DNA degradation (Green and Reed 1998; Liu et al 1997) suggest that in order for ovarian cells to complete the apoptotic program they must contain caspase-3, and an endogenous nuclear DNAase. Boone et.al. (Boone and Yan 1995) demonstrated that granulosa and luteal cells contain endogenous nuclear DNAase, and they hypothesized that these cells would therefore only require a signal to activate this enzyme in order to degrade their DNA in an apoptotic fashion (Laun et al. 2000; Boone and Tsang 1997).

#### **4.4 Conclusion**

In conclusion, we have shown that EMF-exposure causes a large proportion of oocytes in the rat ovary to degenerate by a mechanism similar to apoptosis. This is evident in the EMFexposed group with a large number of degenerative oocytes. Other findings of the study are: an increased number of macrophages; autophagic vacuoles in some granulosa cells; and appearance of several lipid droplets in thecal and luteal cells. The present study has also shown the increased number of macrophages not only in the corpora lutea but also in the growing follicles in the EMF-exposed group. Based on our TEM, we have proposed a model that can explain the initiation of apoptosis by EMF-exposure in ovaries.

The aim of this work was to monitor the reproductive effect of exposure to a magnetic field in rat. Taken together, our results suggest that apoptosis plays a critical role in the degeneration of ovarian cortical tissue, luminal epithelium, glandular epithelium and stromal cells in uterus and luminal epithelium in fallopian tube. The present EMF- exposure model can be used when striving to find ways to improve the viability of ovarian tissue in

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**5** 

*Iraq* 

Batool Mutar Mahdi

**Role of Tumor Marker CA-125 in** 

*Al-Kindy College of Medicine - Baghdad University,* 

**the Detection of Spontaneous Abortion** 

Spontaneous abortion represents a common pregnancy adverse outcome and is a serious emotional burden for women. Loss of pregnancy is a distressing problem for both the

The clinical diagnosis of threatened abortion is presumed when any bloody vaginal discharge or bleeding appears during the first trimester of pregnancy. A prospective study on women with threatened abortion reported that women older than 34 years had an odds ratio of 2.3 for miscarriage (Falco et al., 1996). Some women who bleed in early pregnancy, approximately half of them, will abort (Weiss et al., 2004). Occasionally, bleeding may persist for weeks, and then it becomes essential to decide whether there is any possibility of continuation of the pregnancy or not. The diagnosis of spontaneous abortion currently depends on a combination of ultrasonography and nine hormonal methods including serum human chorionic gonadotropin (HCG), estradiol (E2), estrone, estriol, progesterone, human placental lactogen, cortisol, urine HCG and urine estrogen (Gerhavd and Runnebaum 1984; Zeimet et al., 1998; Osmanagaoglu et al., 2010). Another parameter that could be used as a predictive marker for a spontaneous abortion or subsequent outcome of pregnancy is Cancer Antigen-125 (CA-125). This antigen is a cell surface high molecular weight glycoprotein. It is a mucin like coelomic antigen, which is detected in 80% of non-mucus epithelial carcinomas of ovary. This antigen is secreted from normal tissues, such as coelomic epithelium, amnion and their derivatives including respiratory system, mesenteric organs and epithelium of female genital system (Berek 2002). An increased CA-125 level is due to genital or non-genital origins. Nongenital causes include hepatic diseases, peritonitis, renal failure, breast, colon and lung cancer, and tuberculosis. Genital causes include: pelvic inflammatory diseases, endometriosis, adenomyosis, leiomioma, ectopic pregnancy, endometrial and ovarian

Serum CA-125 levels are increased in early pregnancy and immediately after birth (Cunningham 2005; Speroff and Fritz 2005), implicating the disintegration of the maternal decidua (i.e., blastocyst implantation and placental separation) as a possible source of the tumor marker elevation (Ayaty et al., 2007). There is a cyclic change in the serum concentration of CA-125 in normal menstruating women. It indicates that CA-125 was

**1. Introduction** 

cancer.

patient and physician.

