**3.3 Effect of EMF on uterine tubes**

The uterine tubes act as the site of fertilization and have a critical role in the conduction of zygote to the uterine cavity. Any changes in the structure and or function of uterine tubes would result in tubal pregnancies. The aim of the present study is evaluating the effect of EMF on uterine tubes by examining histological and morphological features using light and electron microscopy and immunohistochemical techniques.

#### **3.3.1 Light microscopic studies**

Light microscopic studies of uterine tubes from control group showed that: uterine tubes are lined with ciliated simple columnar epithelium and their cilia formed a smooth and orderly arranged ribbon at luminal face. The nuclei of epithelial cells were light and euchromatic. The epithelium was rested on a highly vascularized loose connective tissue (Figure 19). In the EMF-exposed group, epithelial cells were low columnar and mostly had lost their cilia. The nuclei of the epithelial cells were condensed and heterochromatic (Figure 20). Morphometric studies, based on final magnification, showed that the height of epithelial cells were decreased after EMF exposure, it was 2.77±0.19 mm VS 2.83±0.46 mm (Figure 21), the difference between two groups is significant (p<0.05).

Environmental Electromagnetic Field and Female Fertility 73

Fig. 21. A diagram showing the height of epithelial cells in uterine tubes (as mm) in control

Electron microscopy showed clearly the ciliated and secretory cells in the epithelial lining of the uterine tubes. In the control group, the ciliated cells had a basal or central ellipsoid nuclei with a prominent nucleolus. The cytoplasm contained poorly developed organells. The apical cell surface bears numerous cilia. The secretory or non ciliated cells had a central elongated nuclei with a prominent nucleolus. The organells were well developed. Membrane bound granules were located at apical cytoplasm. The epithelial cells were held

Fig. 22. Electron micrograph from uterine tube epithelium in control rat. Secretory cell (SC),

together by junctional complex and rested on a basal lamia (Figure 22).

and EMF-exposed groups.

ciliated cell(CC). 3000X.

**3.3.2 Electron microscopic studies** 

Fig. 19. Photomicrograph of uterine tube from control rat. Note epithelium with a ribbonlike cilia at luminal face (arrow). H&E staining. 600X.

Fig. 20. Photomicrograph of a section of uterine tube from EMF-exposed rat. Note loss of cilia in epithelial cells, scattered cilia are left (arrow head). H&E staining. 600X.

Fig. 19. Photomicrograph of uterine tube from control rat. Note epithelium with a ribbon-

Fig. 20. Photomicrograph of a section of uterine tube from EMF-exposed rat. Note loss of

cilia in epithelial cells, scattered cilia are left (arrow head). H&E staining. 600X.

like cilia at luminal face (arrow). H&E staining. 600X.

Fig. 21. A diagram showing the height of epithelial cells in uterine tubes (as mm) in control and EMF-exposed groups.

#### **3.3.2 Electron microscopic studies**

Electron microscopy showed clearly the ciliated and secretory cells in the epithelial lining of the uterine tubes. In the control group, the ciliated cells had a basal or central ellipsoid nuclei with a prominent nucleolus. The cytoplasm contained poorly developed organells. The apical cell surface bears numerous cilia. The secretory or non ciliated cells had a central elongated nuclei with a prominent nucleolus. The organells were well developed. Membrane bound granules were located at apical cytoplasm. The epithelial cells were held together by junctional complex and rested on a basal lamia (Figure 22).

Fig. 22. Electron micrograph from uterine tube epithelium in control rat. Secretory cell (SC), ciliated cell(CC). 3000X.

Environmental Electromagnetic Field and Female Fertility 75

Immunohistochemical studies were restricted to TUNEL technique for detection of apoptotic cells. In the control group, with TUNEL assay, apoptotic cells was neither observed in the lining epithelium nor in the subepithelial layer (Figure 24). In the EMFexposed group, there were numerous apoptotic cells in the lining epithelium. Apoptosis was observed both in secretory and ciliated cell types. Very few apoptotic cells was also present

Fig. 24. Photomicrograph of a section from uterine tube in control rat. TUNEL method,

counterstained with toluidine blue. 360X.

**3.3.3 Immunohistochemical studies** 

in the subepithelial layer (Figure 25).

In the EMF-exposed group, the nuclei were condensed, the apical cilia were apparently reduced and organells were sparsely distributed through the cytoplasm. Slight pyknosis, increased peripheral chromatin condensation and some degree of cytoplasmic condensation was observable. The most remarkable feature of the uterine tubes in this group was the presence of cells with preapoptotic characteristics i.e. flattened and cystic endoplasmic reticulum and nuclear condensation (Figure 23).

Fig. 23. Electron micrograph from uterine tube in EMF-exposed rat. Note decreased ciliar number and presence of intercellular spaces and cystic rER. 3500X.

#### **3.3.3 Immunohistochemical studies**

74 From Preconception to Postpartum

In the EMF-exposed group, the nuclei were condensed, the apical cilia were apparently reduced and organells were sparsely distributed through the cytoplasm. Slight pyknosis, increased peripheral chromatin condensation and some degree of cytoplasmic condensation was observable. The most remarkable feature of the uterine tubes in this group was the presence of cells with preapoptotic characteristics i.e. flattened and cystic endoplasmic

Fig. 23. Electron micrograph from uterine tube in EMF-exposed rat. Note decreased ciliar

number and presence of intercellular spaces and cystic rER. 3500X.

reticulum and nuclear condensation (Figure 23).

Immunohistochemical studies were restricted to TUNEL technique for detection of apoptotic cells. In the control group, with TUNEL assay, apoptotic cells was neither observed in the lining epithelium nor in the subepithelial layer (Figure 24). In the EMFexposed group, there were numerous apoptotic cells in the lining epithelium. Apoptosis was observed both in secretory and ciliated cell types. Very few apoptotic cells was also present in the subepithelial layer (Figure 25).

Fig. 24. Photomicrograph of a section from uterine tube in control rat. TUNEL method, counterstained with toluidine blue. 360X.

Environmental Electromagnetic Field and Female Fertility 77

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

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

events

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

apoptosis (Payne et al. 1995).

apoptosis.

**4.2 The unique aspects of oocytes** 

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 blue. 360X.
