**3.1.1 Light microscopic studies**

58 From Preconception to Postpartum

breast cancer, kidney cancer and or cardiovascular disease (Kovacic and Pozos 2006; NRC 1996; UNEP/WHO/IRPA 1987). An ultra-structural study on rats shows that long term exposure to EMF could result in lymphatic organ disturbances and consequently weakening of immune system ( Mohammadnejad et al. 2010). Some authors have reviewed risks of EMF exposure on reproduction and demonstrated that experimental exposure to EMF in laboratory animals has several adverse effects (Djeridane et al. 2008; Ozguner et al. 2005; Chung et al. 2005; Ahmed et al. 2002; elbetieha et al. 2002; Cheronff et al. 1992). There are numerous studies showing that EMF exposure of male rat/mice affects testicular architecture , spermatogenesis, sperm motility, leydig cell reduction, increased apoptosis of germ cells and in general subfertility and or infertility ( Khaki et al. 2006; Lee et al. 2004; Soleimani Rad and Katebi 1997; Devita et al. 1995; Lokmatova 1993). In human and animal studies, it has been reported that female exposure to EMF cause some adverse effects and reviewed the potential effect of EMFs on infertility, implantation rate, number of living fetuses, sex ratio, miscarriages, premature births, growth retardation, low birth weight, congenital malformations and prenatal deaths (Roshangar and Soleimani Rad 2007; Lahijani et al. 2007; Feychting et al. 2005; Chiang et al. 1995; Huuskonen et al. 1993; Juutilainen et al.

Although the epidemiological studies on the effect of EMF exposure in human, are not conclusive but the experimental findings on the effect of EMF on different organs would act as a corner stone for exploring the complicated topic of EMF effect on reproduction. As several organs are involved in the fertility of females and their well being are necessary for fertility, the present chapter will deal with the effect of EMF on three major female

In these series of experiments the Wistar rats are used as an experimental model and the studies were approved by the ethical committee of Tabriz University of Medical Sciences. For producing EMF, an EMF generating apparatus was designed in the Department of the Histology and Emberyology. The apparatus uses 220 V and 50 HZ alternative current and could generate up to 5 milli tesla EMF. In the presented studies, the adult female rats were exposed to 3 milli tesla, 4 hours/day for 2 months. After the experimental period the rats were sacrificed and their reproductive organs including; ovaries, uteri, and uterine tubes were dissected apart. Half of the samples were fixed in 10% formalin and paraffinembedded sections were used for light microscopy and immunohistochemical studies. TUNEL techniques were used for detection of apoptosis and Ki-67 technique for

The other half of the samples were fixed in 2% glutaraldehyde and resin embedded ultra

Ovary is an organ involved in follicular development and germ cell production. In other word, the ovary provides a proper environment for oogenesis. Any disturbances in ovarian

proliferation assay. In both cases toluidine blue was used for counterstaining.

1993; Mc Govern et al. 1990).

**2. Methods** 

**3. Results** 

reproductive organs i.e. ovary, fallopian tubes and uterus.

al.

thin sections were used for electron microscopy.

**3.1 Ovarian effect of EMF exposure** 

Light microscopy showed that, in control group, oocyte had a euchromatic nucleus and was encompassed with a homogenous zona pellucida, and well organized corona radiate and granulosa cells (Figure 1-A). In experimental group, the oocyte had a condensed nucleus so that it appeared small and darkly stained ( Figure 1B). The cytoplasm of the oocytes were condensed and surrounding zona pellucida had changed and appeared narrower than that in control group (Figure 1B). The cells in granulosa and corona radiata layers were disorganized and contained dense nuclei (Figure 1B).

Fig. 1. Photomicrographs of ovarian sections from rat ovaries. A-from control group, showing part of a graffian follicle with oocyte and cumulus. B-from EMF-exposed group, showing an oocyte with condensed nucleus, surrounded with a faint zona pellucida and an irregularly arranged cumulus and corona radiata. H&E staining. 300X.

Ovarian stroma both in cortex and medulla contained several macrophages wich was rarely seen in control group and their number was higher in cortex than the medulla (Figure 2). Morphometric studies revealed that the number of ovarian follicles, in different stages of development, were higher in EMF-exposed group, but the number of corpora lutea were fewer than the control group. Atretic follicles in the EMF-exposed group were numerous than the control group.

#### **3.1.2 Electron microscopic studies**

Ultra structural studies revealed that granulosa cells in control group, were regularly arranged and the corona radiate layer was composed of columnar cells that were attached to eachother by intercellular junctions. Their microvilli were penetrated into zona pellucid and could be recognized in it. The cytoplasm of coronal cells contained different organels including spherical or ovoid mitochondria with limited cristae (Figure 3).

Environmental Electromagnetic Field and Female Fertility 61

Cytoplasm of oocytes in this group contained numerous lamellae, scattered organells including round or ovoid mitochondriae with short and limited crista. The endoplasmic reticulum were poorly developed (Figure 4). The nuclei of oocytes were euchromatic and contained an obvious nucleolus. In the vicinity of granulosa cell layer the capillary-rich

Ovary also contained several corpora lutei composed of large and round cells with a prominent nuclei and nucleoli. The luteal cells were pale and contained numerous mitochondria, an extensive rough endoplasmic reticulum, smooth endoplasmic reticulum and Golgi apparatus. The cells also contained numerous free ribosomes, lipid droplets and a

In EMF-exposed group, the zona pellucida was narrower than in control, 3.24±0.25 µm VS 4.47±0.42 µm, and the difference was significant (p<0.001). The number of microvilli profiles per unit area in this group was fewer than in control, 4.13±0.83 VS 9.8±0.56, which was statistically significant (p<0.001) and (Figure 7).The coronal cells were shrunken, separated

Fig. 4. Electron micrograph of cytoplasm of oocyte from a control rat ovary. Note

mitochondria (M), lamellae (L), and secondry lysosome (SL). 15000 X.

theca interna and outer to it the collagen-rich theca externa were present.

few number of lysosomes and multivesicular bodies (Figure 5).

from zona pellucid and lost contact from eachother (Figure 6).

Fig. 2. Photomicrograph of a section of an ovary from EMF-exposed rat. Showing several macrophages with brownish residual bodies (\*). H&E staining. 200X.

Fig. 3. Electron micrograph of developing follicle from a control rat ovary. Note the thickness of zona pellucida and sections of microvilli from oocyte and granulosa cells in it. cellular junction between granulosa cells is obvious (arrow). 15000X.

Fig. 2. Photomicrograph of a section of an ovary from EMF-exposed rat. Showing several

Fig. 3. Electron micrograph of developing follicle from a control rat ovary. Note the thickness of zona pellucida and sections of microvilli from oocyte and granulosa cells in it.

cellular junction between granulosa cells is obvious (arrow). 15000X.

macrophages with brownish residual bodies (\*). H&E staining. 200X.

Cytoplasm of oocytes in this group contained numerous lamellae, scattered organells including round or ovoid mitochondriae with short and limited crista. The endoplasmic reticulum were poorly developed (Figure 4). The nuclei of oocytes were euchromatic and contained an obvious nucleolus. In the vicinity of granulosa cell layer the capillary-rich theca interna and outer to it the collagen-rich theca externa were present.

Ovary also contained several corpora lutei composed of large and round cells with a prominent nuclei and nucleoli. The luteal cells were pale and contained numerous mitochondria, an extensive rough endoplasmic reticulum, smooth endoplasmic reticulum and Golgi apparatus. The cells also contained numerous free ribosomes, lipid droplets and a few number of lysosomes and multivesicular bodies (Figure 5).

In EMF-exposed group, the zona pellucida was narrower than in control, 3.24±0.25 µm VS 4.47±0.42 µm, and the difference was significant (p<0.001). The number of microvilli profiles per unit area in this group was fewer than in control, 4.13±0.83 VS 9.8±0.56, which was statistically significant (p<0.001) and (Figure 7).The coronal cells were shrunken, separated from zona pellucid and lost contact from eachother (Figure 6).

Fig. 4. Electron micrograph of cytoplasm of oocyte from a control rat ovary. Note mitochondria (M), lamellae (L), and secondry lysosome (SL). 15000 X.

Environmental Electromagnetic Field and Female Fertility 63

The cytoplasm of oocytes showed fewer organell clusters and contained higher number of lamellae. The rough endoplasmic reticulum were poorly developed, mitochondria were smaller and numerous fat droplets were present in comparison to control group (Figure 7).

Numerous granulosa cells near the antrum had typical characteristics of apoptotic cells. They had condensed and cresent like nuclei and were separated from neighboring cells (Figure 9). The granulosa cells had also irregular basal lamina and in some follicles were broken. The thecal cells, in comparison to control group, had more condensed nuclei and contained; several lipid droplets, autophagic granules, apoptotic bodies, ruptured mitochondria, dilated nuclear membrane and cytoplasmic vesiculation (Figure 9). Similar changes were observed in the stromal cells from EMF-exposed group. In addition, several macrophages containing apoptotic bodies and multivesicular bodies were also present (Figure 10). These cells were mainly located near the blood vessels. The cells with cresent like nuclei, which is the characteristics of apoptotic cells, were present

Fig. 7. Electron micrograph of cytoplasm of oocyte from EMF-exposed rat. Secondary

lysosome (SL), lamellae (L), and mitochondria (M). 15000 X.

The nuclei had irregular counter and nucleolus were condensed (Figures 8).

among stromal cells.

Fig. 5. Electron micrograph of corpus luteum from a control rat ovary. Note normal organels and some lipid droplets (L). 5000X.

Fig. 6. Electron micrograph of an oocyte with zona pellucida and granulosa cells from EMFexposed rat. Note condensed granulosa cells that lost contact from zona pellucida and neighboring cells (GC). Zona pellucid (ZP), and oocyte (O). 3000X.

Fig. 5. Electron micrograph of corpus luteum from a control rat ovary. Note normal organels

Fig. 6. Electron micrograph of an oocyte with zona pellucida and granulosa cells from EMFexposed rat. Note condensed granulosa cells that lost contact from zona pellucida and

neighboring cells (GC). Zona pellucid (ZP), and oocyte (O). 3000X.

and some lipid droplets (L). 5000X.

The cytoplasm of oocytes showed fewer organell clusters and contained higher number of lamellae. The rough endoplasmic reticulum were poorly developed, mitochondria were smaller and numerous fat droplets were present in comparison to control group (Figure 7). The nuclei had irregular counter and nucleolus were condensed (Figures 8).

Numerous granulosa cells near the antrum had typical characteristics of apoptotic cells. They had condensed and cresent like nuclei and were separated from neighboring cells (Figure 9). The granulosa cells had also irregular basal lamina and in some follicles were broken. The thecal cells, in comparison to control group, had more condensed nuclei and contained; several lipid droplets, autophagic granules, apoptotic bodies, ruptured mitochondria, dilated nuclear membrane and cytoplasmic vesiculation (Figure 9). Similar changes were observed in the stromal cells from EMF-exposed group. In addition, several macrophages containing apoptotic bodies and multivesicular bodies were also present (Figure 10). These cells were mainly located near the blood vessels. The cells with cresent like nuclei, which is the characteristics of apoptotic cells, were present among stromal cells.

Fig. 7. Electron micrograph of cytoplasm of oocyte from EMF-exposed rat. Secondary lysosome (SL), lamellae (L), and mitochondria (M). 15000 X.

Environmental Electromagnetic Field and Female Fertility 65

Fig. 10. Electron micrograph of ovarian stroma from an EMF-exposed rat. Note necrotic

Immunohistochemical studies was performed on paraffin sections using TUNEL technique for detection of apoptosis and Ki-67 technique for proliferation assay. Both TUNEL positive and Ki-67 positive cells were distinct from nonpositive cells by their brownish color. In control group TUNEL positive cells were fewer and was limited to granulosa cells near the antrum of atretic follicles (Figure 11). Proliferative cells (Ki-67 positive cells), in this group,

In experimental group, exposed to EMF, apoptotic cells were mainly found near the antrum but also were present among cells close to basement membrane (Figure 12). TUNEL positive cells were not observed in thecal layers but were observed in ovarian stroma. The corpora lutea in the EMF-exposed group also contained numerous TUNEL positive cells. The result from apoptotic cells indicate that EMF exposure induces apoptosis not only in granulose cells but also in other parts of the ovary. Regarding Ki-67 assay, the number of Ki-67 positive cells in granulosa layer from EMF-exposed group were obviously fewer than that in

In experimental group, exposed to EMF, apoptotic cells were mainly found near the antrum but also were present among cells close to basement membrane (Figure 12). TUNEL positive cells were not observed in thecal layers but were observed in ovarian stroma. The corpora lutea in the EMF-exposed group also contained numerous TUNEL positive cells. The result from apoptotic cells indicate that EMF exposure induces apoptosis not only in granulose cells but also in other parts of the ovary. Regarding Ki-67 assay, the number of Ki-67 positive cells in granulosa layer from EMF-exposed group were obviously fewer than that in

cells, and macrophages containing multivesicular and apoptotic bodies. 3700X.

were observed in granulose layer close to the basement membrane.

control group meaning that EMF exposure inhibits proliferation.

control group meaning that EMF exposure inhibits proliferation.

**3.1.3 Immunohistochemical studies** 

Fig. 8. Electron micrograph of oocyte nucleus and cytoplasm from an EMF-exposed rat. Note nucleus and nucleoulus. 10000 X.

Fig. 9. Electron micrograph of a developing follicle from EMF-exposed rat. Apoptotic cell (A), basal lamina (arrow). 3000X.

Fig. 8. Electron micrograph of oocyte nucleus and cytoplasm from an EMF-exposed rat.

Fig. 9. Electron micrograph of a developing follicle from EMF-exposed rat. Apoptotic cell

Note nucleus and nucleoulus. 10000 X.

(A), basal lamina (arrow). 3000X.

Fig. 10. Electron micrograph of ovarian stroma from an EMF-exposed rat. Note necrotic cells, and macrophages containing multivesicular and apoptotic bodies. 3700X.
