Evaluation of the effects of extremely low frequency electromagnetic fields on mammalian follicle development

Sandra Cecconi1,4, Giancaterino Gualtieri1, Angela Di Bartolomeo1, Giulia Troiani1, Maria Grazia Cifone2 and Rita Canipari3

1 Department of Biomedical Sciences and Technologies, 2 Department of Experimental Medicine, University of L'Aquila and 3 Department of Histology and Medical Embryology, University `La Sapienza', Rome


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to evaluate the effects of pulsed, extremely low-frequency electromagnetic fields (ELF–EMF) on in-vitro mouse pre-antral follicle development. Pre-antral follicles were cultured for 5 days and exposed to ELF–EMF at the frequencies of 33 or 50 Hz. ELF–EMF application did not affect follicular growth over a 3 day culture period, but on day 5 the growth of 33 Hz-exposed follicles was significantly reduced when compared with controls, while the 50 Hz-exposed follicles were not significantly affected. However, ELF–EMF severely impaired antrum formation at both frequencies, as 79 ± 3% of control follicles developed antral cavities compared with 30 ± 6% and 51.6 ± 4% of 33 or 50 Hz-exposed follicles respectively. The follicles with failed antrum formation showed lower oestradiol release and granulosa cell DNA synthesis, but these effects were not related to granulosa cell apoptosis. Furthermore, a high percentage of the in-vitro grown oocytes obtained from exposed follicles had a reduced ability to resume meiotic maturation when compared with controls. These results suggest that ELF–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.

Key words: apoptosis/extremely low frequency electromagnetic field/meiotic maturation/pre-antral follicles


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The increasing use of electric power for domestic and industrial appliances has resulted in the exposure of many millions of daily users, in homes and workplaces, to a complex mix of artificially elevated electromagnetic fields (EMF) that span a wide frequency range.

The effects of extremely low-frequency electromagnetic fields (ELF–EMF; <=100 Hz) on the biological functions of living organisms represent an emerging area of interest for human health. Data reported in the literature regarding direct effects exerted by ELF–EMF on cell functions are somewhat controversial (McCann et al., 1998Go) since ELF–EMF exposure is considered as `global interference or stress to which a cell can adapt without catastrophic consequences' (Goodman et al., 1995, for review). However, an increasing number of reports indicate that these magnetic fields are involved in cancer induction as co-carcinogenic factors capable of enhancing the effects of other mutagenic substances (Baum et al., 1995Go; Mevissen et al., 1995Go). Indeed, the application of a wide range of field intensities (3–100 Hz) can negatively affect haemopoiesis (Bonhomme-Faivre et al., 1998Go; Mevissen et al., 1998Go), enhance growth rates of transformed cells in some human epithelial cancers (Watson et al., 1998Go), and even reduce the effect of antineoplastic drugs (Harland and Liburdy, 1997Go). No genotoxic effects, in terms of DNA damage, have been reported for ELF–EMF-exposed cells (Fiorani et al., 1992Go; Morandi et al., 1996Go; McCann et al., 1998Go; Scarfi et al., 1999Go) even though increased amounts of mRNA and proteins of specific genes, such as c-myc, c-jun, c-fos, heat shock proteins (HSP) 70 and hypoxanthine–guanine phosphoribosyltransferase (HGPT) have been described for various cell types (Mather et al., 1990Go; Miyakoshi et al., 1997Go; Campbell-Beachler et al., 1998Go; Han et al., 1998Go; Jahreis et al., 1998Go; Lagroye and Poncy, 1998Go; Lin et al., 1998Go; Loberg et al., 1999Go). Various reports suggest that the interaction site for ELF–EMF is the plasma membrane, since exposure determines altered Ca2+ influx (Conti et al., 1985Go; Liburdy, 1992Go; Flipo et al., 1998Go; Fanelli et al., 1999Go; Walleczek et al., 1999Go), as well as the clustering of integral plasma membrane proteins (Bersani et al., 1997Go). In addition, ELF–EMF-induced oscillations of intracellular Ca2+ concentration (Loschinger et al., 1998Go) have been correlated with changes in DNA, RNA and protein synthesis activities (Liburdy, 1992Go). More recently, the application of ELF–EMF has been involved in the reduction (Fanelli et al., 1999Go) or stimulation (Flipo et al., 1998Go; Ismael et al., 1998Go) of somatic cell apoptosis. In both cases, the apoptotic process appears to be modulated by Ca2+-dependent mechanisms.

There is very little information in the literature regarding the possible harmful effects of ELF–EMF on the reproductive system. One report by Denegre et al. (1998) demonstrates that exposure to a very strong static magnetic field (1T) can alter normal cleavage planes of Xenopus embryos, thus suggesting a direct action on the microtubles of the mitotic apparatus. In mammals, the application of lower frequency fields (50 Hz, 1–100 mT) can affect the proliferative/differentiative capacity of mouse spermatogonia (Furuya et al., 1998Go), but does not induce clastogenic effects on human sperm chromosomes (Tateno et al., 1998Go). At present, the only report on mammalian oocytes, by Mailhes and colleagues (1997), demonstrates that electromagnetic fields enhance chemically-induced hyperploidy in mouse oocytes. However, no other data concerning the role played by ELF–EMF on important aspects of mammalian oogenesis are available. As a consequence, this study was designed to evaluate whether ELF–EMF could affect mouse ovarian follicle development. Several morphological and biochemical parameters representative of physiological follicle development were tested, including follicle growth, antrum formation, oestradiol release, granulosa cell apoptosis and oocyte meiotic maturation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Swiss CD1 mouse families (Charles River, Como, Italy) were housed in a temperature- (22–23°C) and light-controlled room (12 h lighting schedule, from 0600–1800 h). Animals were maintained in accordance with the principles outlined in the National Institute of Health (NIH) guide for the care and use of laboratory animals. When needed, 22 day old females received 5 IU pregnant mare serum gonadotrophin (PMSG) 48 h before mating (PMSG-primed mice).

Chemicals
All chemicals used were purchased from Sigma Chemical (St Louis, MO, USA). Highly purified ovine follicle stimulating hormone (FSH)(National Institute of Diabetes, Digestive and Kidney Disease, NIDDK-o-FSH-19-SIAFP,BIO) was kindly provided by the National Hormone and Pituitary Program of the NIH.

Pulsed ELF–EMF exposure system
The ELF–EMF used in the present study were produced by a pair of Helmholtz coils able to generate a highly homogeneous field (with a homogeneity >5/1000) over 64 wells of a 96-well plate. The power pulse generator built for the Helmholtz coils was able to generate an effective magnetic field in the range 0–2.8 mT (0–28 G), with a square wave from 1–75 Hz. Computation of the field distribution and homogeneity were carried out using a Laplace equation simulation programme that takes into account the finite dimension of the coil. Field intensity was measured with a DTM-141 digital teslamer (group 3, Danfysin, Wellington, New Zealand) equipped with an LPT-141-125 hall probe. The field intensity for both frequencies was fixed to 1.50 ± 0.01 mT (15.0 ± 0.1 G). The plate was positioned in the central region of the Helmoltz coil, not in contact with the culture plates to avoid any temperature increase due to the Joule effect in the coils. To exclude uncontrolled thermal effects of the field during culture, the maintenance of 37 ± 0.1°C inside each exposed well was controlled by direct temperature measurement with thermocouple. In this study, two different pulsed square waves at frequencies of 33 and 50 Hz, with 50% of duty cycle, were chosen to expose cells. The square wave was utilized because it contained the fundamental frequency (33 or 50 Hz) and all their odd harmonics (multiple frequencies) with their relative weights. The released energy was directly related to duty cycle waveform, and at 50% duty cycle only the fundamental frequency ({nu}), the first (3v) and the second (5v) harmonics were very strong. In this way, a possible effect due to 33, 99 and 165 Hz, or 50, 150 and 250 Hz frequencies was tested.

Pre-antral follicle isolation and culture
Individual pre-antral follicles were mechanically dissected from ovaries of 22 day old mice using fine needles. All follicles were isolated with a small clump of thecal stromal tissue attached and were measured with a precalibrated ocular micrometer at x40 magnification. Only those measuring 160 ± 10 µm in diameter (excluding thecal tissue), with a centrally placed spherical oocyte (mean diameter: 69 ± 1 µm), and with no signs of somatic cell degeneration were chosen for further culture. Follicles were individually placed in 96-V-well microtitre plates in 25 µl alpha minimal essential medium ({alpha}MEM) supplemented with 1% ITS (insulin 10 ng/ml; transferrin 5.5 ng/ml; selenium 5 ng/ml), antibiotics (penicillin, 100 IU/ml; streptomycin, 100 mg/ml), 100 ng/ml ovine FSH, and 5% fetal calf serum (FCS). The medium was overlaid with 70 µl sterile mineral oil (embryo tested, density 0.84 g/ml). Follicles were cultured for 5 days at 37°C in a 5% CO2 atmosphere and exposed or not exposed (controls) to ELF–EMF. The medium in each well was replaced every day, and collected samples were stored at –80°C for measurement of 17ß-oestradiol.

Evaluation of follicle and oocyte growth and development
Follicle morphology was observed under inverted microscope, and follicle as well as oocyte diameters were recorded daily. At the end of the culture period, the formation of an antral cavity was determined by the presence of a visible translucent area inside the follicle. Follicles were carefully opened and the oocytes, mechanically isolated from surrounding cumulus cells, were measured with a precalibrated ocular micrometer at x40 magnification and further incubated for 16–18 h in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.23 mmol/l sodium pyruvate, 5% FCS and 100 ng/ml ovine FSH to allow resumption of meiosis. At the end of the maturation period, oocytes were analysed for meiotic resumption.

DNA content was determined to verify if expansion in follicle size reflected an increase in the cell number. DNA extracted from pools of six follicles per assay at day 0 and 5 of culture was measured by the fluorometric assay using Hoechst 33258 (0.1 mg/ml) as a fluorescent dye. Aliquots of samples were added to 2 ml of dye solution and immediately measured by a fluorometer (Perkin-Elmer, Milan, Italy) at 365/460 nm (excitation/emission) wavelengths. DNA was expressed as ng/follicle, and dilutions of sonicated salmon sperm DNA were used as a standard.

17 ß-oestradiol determination
The concentration of 17ß-oestradiol in the medium was determined by a radioimmunoassay kit according to the procedure described by the manufacturer (Radim, Pomezia, Italy) as previously reported (Manna et al., 1991Go). The intra-assay and the interassay coefficients of variation were 4 and 5% respectively. Results from radioimmunoassay were analysed with a program that uses a four-parameter logistic function, and unknowns were interpolated from the resultant curve.

Morphological analysis of granulosa cell apoptosis
Intact pre-antral follicles (160 ± 10 µm) and early antral follicles (300±10 µm), obtained from ovaries of 22 day old mice, were cultured in 25 µl serum-free {alpha}MEM supplemented with antibiotics (penicillin, 100 IU/ml; streptomycin, 100 mg/ml) and 0.1% bovine serum albumin (BSA) under oil, and exposed or not exposed (controls) to a 33 Hz frequency. Granulosa cells were released after mechanical dissection of these follicles before (time = 0) and after 24 and 48 h of culture to determine the incidence of apoptosis. Pools of granulosa cells (eight follicles per assay) were fixed for 15 min in 4% neutral buffered formalin in phosphate buffered saline (PBS) pH 7.4 and cytocentrifuged onto a glass slide at 200 g for 10 min. The samples were washed three times with PBS, the chromatin stained for 10 min at room temperature with Hoechst 33258 (0.1 mmol/l) and examined by fluorescent microscopy or with the TUNEL (TdT-mediated dUTP-X nick end labelling) method according to the manufacturer's procedures (Apop TagTM, #S7100-Kit, Oncor, Gaithersburg, MD, USA). The apoptotic bodies were identified and counted in three or more randomly selected fields of at least 100 cells each, at x1000 magnification. The same experimental protocol was applied to pre-antral granulosa cells obtained from in-vitro grown follicles stained at the end of culture on day 5.

Apoptosis evaluation by propidium iodide solution
Apoptosis was further assayed on dispersed early antral granulosa cells by flow cytometry (Nicoletti et al., 1991Go). Granulosa cells obtained by puncturing ovaries of 22 day old mice were either cultured in DMEM supplemented with 0.1% BSA for 24–48 h as control, or exposed to a 33 Hz frequency, or immediately treated (time = 0) for apoptosis detection. About 3x105 viable cells, as judged by Trypan Blue dye exclusion test, were gently resuspended in 1.5 ml hypotonic propidium iodide solution (PI, 50 µg/ml in 0.1% sodium citrate/0.1% Triton X-100) and kept overnight in the dark at 4°C. The PI-fluorescence of individual nuclei was measured by flow cytometry with standard FACScan equipment (Becton Dickinson, Mountain View, CA, USA). The nuclei traversed the light beam of a 488 nm argon laser. A 560 nm dichroid mirror (DM 570) and a 600 nm band pass filter (band width 35 nm) were used to collect the red fluorescence due to PI DNA staining, and the data were recorded on a logarithmic scale in a Hewlett Packard (HP 9000, model 310, Palo Alto, CA, USA) computer. The percentage of apoptotic cell nuclei (sub-diploid DNA peak in the DNA fluorescence histogram) was calculated with FACScan research software Lysis II.

Cumulus cell–oocyte complex isolation and culture
Ovaries from PMSG-primed mice were excised and placed in a HEPES-buffered medium (Quinn et al., 1982Go). Cumulus–oocyte complexes (COCs) were released by puncturing with a needle large pre-ovulatory follicles (>400 µm in diameter). COCs were transferred to a 100 µl drop of DMEM supplemented with 0.23 mmol/l sodium pyruvate, 5% FCS and 100 ng/ml ovine FSH, and exposed or not exposed (controls) for 16–18 h to 33 or 50 Hz fields. At the end of the culture, cumulus expansion was evaluated by morphological and physical criteria using a stereomicroscope. Cumulus cell dispersion and embedding in the matrix with hyaluronic acid were considered as positive signs of mucification. COCs were then digested with 300 mg/ml hyaluronidase and oocytes were recovered for examination of meiotic resumption.

Analysis of meiotic resumption
The stage of oocyte meiotic maturation was assessed by Hoechst 33342 staining (5 µg/ml). Meiotic arrest was indicated by the presence of germinal vesicle (GV) and nucleolus, while breakdown of these nuclear structures (GVBD) and the appearance of the first polar body (PB) served as markers for resumption of meiosis and oocyte maturation.

Statistical analysis
Statistical analysis was performed by analysis of variance (ANOVA) followed by the Tukey–Kramer test for comparison of multiple groups.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of ELF-EMF exposure on in-vitro follicle development, oestradiol release and granulosa cell proliferation
The effects exerted on pre-antral follicle development by pulsed ELF–EMF at the two selected frequencies of 33 and 50 Hz was evaluated during a 5 day culture period. Under control conditions, pre-antral follicles (160 ± 10 µm; Figure 1aGo) grew rapidly, and on day 5 the majority (79 ± 3%) reached the large pre-ovulatory stage (about 400 µm; Figure 1bGo) characterized by a fully-expanded antral cavity (Figure 1cGo). Pre-antral follicles exposed to ELF–EMF and unexposed controls grew, by the third day of culture, to a similar extent, reaching a diameter of 260 ± 10 µm and 290 ± 10 µm respectively. From day 4 onward, the growth rate of the 33 Hz-exposed follicles decreased significantly, and by the end of culture their mean follicle diameter was 76% that of controls (P < 0.01). The growth rate of 50 Hz-exposed follicles decreased slightly when compared with controls, though not significantly (Figure 2AGo). However, ELF–EMF exposure at both frequencies reduced the proportion of follicles capable of further development. In fact, by day 5, only 30 ± 6% (P < 0.001) and 51.6 ± 4% (P < 0.01) of 33 and 50 Hz-exposed follicles respectively showed the presence of large antral cavities compared with 79 ± 3% of control follicles (Figure 2BGo). On the basis of these results, follicles were divided into two different groups, with or without antrum, before any further evaluation.



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Figure 1. Photomicrographs of: (a) freshly isolated mouse pre-antral follicle; follicles cultured in the presence of ovine FSH as described and showing (b) the absence or (c) the presence of a well-developed antral-like cavity on day 5 of culture. Bar represents 100 µm.

 


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Figure 2. (A) Growth rate of follicles exposed or not exposed to 33 and 50 Hz frequencies for 5 days; (B) percentage of antral cavities shown by mouse pre-antral follicles on day 5 of culture. Values represent the mean ± SEM of five independent experiments. *P < 0.01, **P < 0.001.

 
In order to determine whether the increase in follicle volume was dependent on cell proliferation, the DNA content per follicle was evaluated. The follicles exposed to 33 Hz that had not developed antral cavities by day 5 of culture presented lower amounts of DNA than controls with antrum (P < 0.01). A correlation between antrum formation and DNA content was demonstrated by the finding that the few follicles that developed antra showed DNA quantities not statistically different from those of controls with antrum (Figure 3AGo). Similar results were obtained from 50 Hz-exposed follicles, though a lower proportion of follicles was negatively affected by exposure at this specific frequency (not shown).



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Figure 3. Changes in: (A) DNA content; and (B) 17ß-oestradiol secretion (*P < 0.05; **P < 0.01 versus control, C + antrum) in follicles cultured for 5 days exposed or not to 33 Hz frequency. Values are the mean ± SEM of five independent experiments. There are no significant differences between groups with the same superscript, while groups with different superscripts are significantly different.

 
The pattern of 17ß-oestradiol production mirrored follicle growth rate. Control and exposed follicles produced similar quantities of 17ß-oestradiol up to the third day of culture (about 2.5 ng/ml). From day 4 onward, steroid production increased in control follicles in concomitance with antrum formation. A similar increase was not observed in the 33 Hz-exposed follicles without antrum, while the few follicles that did develop antra showed 17ß-oestradiol levels not statistically different from those of controls (Figure 3BGo).

Effect of ELF–EMF exposure on follicle cell apoptosis
In order to determine whether ELF–EMF could impair follicle growth by affecting granulosa cell apoptosis, the presence of apoptotic bodies was evaluated in 33 Hz-exposed and unexposed pre-antral follicles. To this end, granulosa cells were stained with Hoechst 33258 or with TUNEL at the end of the 5-day culture period. It was found that similar low proportions of granulosa cells showed morphological signs of apoptosis (about 8%), without significant differences between treatments. A second series of experiments evaluated whether and, if so, to what extent ELF–EMF exposure to 33 Hz could affect apoptosis of granulosa cells obtained from follicles at different developmental stages. Spontaneous granulosa cell death was experimentally induced by culturing follicles in serum-free medium (McGee et al., 1997Go). The proportion of apoptotic bodies in granulosa cells obtained from freshly harvested (time = 0) and 24–48 h exposed or unexposed intact pre-antral and early antral follicles was evaluated by Hoechst 33258 and TUNEL stainings. The results show that no significant induction was attributable to field exposure. In fact, as shown in Figure 4Go, the percentage of pre-antral and early antral granulosa cells presenting the nuclear morphological changes characteristic of apoptosis increased during culture time, but the values were similar in both exposed and unexposed cells. To confirm the morphological results, a quantitative assay of DNA content on dispersed early antral granulosa cells was performed by flow cytometry. The results showed that apoptosis increased with time, though no specific effect was due to the prolonged ELF–EMF exposure (data not shown).



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Figure 4. Detection of apoptosis in granulosa cells obtained from control or 33 Hz-exposed pre-antral or early antral follicles cultured in serum-free medium for increasing length of time. Upper panel: representative fluorescence micrographs of Hoechst staining of pre-antral granulosa cells at time = 0 h (a), or cultured for 48 h exposed (b) or not exposed (c) to 33 Hz frequency. Apoptotic nuclei are evidenced by chromatin clumping (*). Lower panel: percentage of pre-antral and early antral granulosa apoptotic cells in the total cell population at 0 and 24 and 48 h of culture. Values are the mean ± SEM of three independent experiments.*P < 0.05, **P < 0.001 versus respective time = 0 h.

 
Effect of ELF–EMF exposure on growth and meiotic competence of oocytes from in-vitro grown follicles
In order to evaluate whether ELF–EMF could affect oocyte growth and competence to mature, oocytes from in-vitro grown follicles were collected on day 5 of culture, their final diameter was measured and they were then allowed to mature in vitro. Under all the experimental conditions tested, oocyte diameters increased significantly from 69 ± 1 to 74 ± 1 µm (P < 0.01), but their ability to resume and complete meiosis was strongly conditioned by ELF–EMF exposure. In fact, the percentages of GVBD and metaphase II (MII) in oocytes obtained from 33 and 50 Hz-exposed follicles without antra were significantly lower than in oocytes from control follicles (Figure 5Go). Conversely, nuclear maturation in oocytes obtained from the few follicles whose antral cavity formation was not affected by ELF–EMF exposure was comparable to the control.



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Figure 5. Maturation competence of oocytes obtained from control or exposed follicles cultured for 5 days. At the end of culture period, oocytes derived from follicles with (+) or without (–) antral cavities were separated and subjected to in-vitro maturation for 17 h. Values are the mean ± SEM of five independent experiments. Number in brackets refers to the percentage of oocytes that underwent germinal vesicle breakdown (GVBD) and exhibited a polar body. *P < 0.05; **P < 0.01 versus respective follicles with antrum as well as versus control follicles.

 
Effect of ELF–EMF on cumulus cell mucification and meiotic maturation in in-vivo grown oocytes
These experiments evaluated whether ELF–EMF exposure could affect the capacity of fully-differentiated COCs to undergo normal cumulus expansion and to complete meiotic maturation up to MII. To this end, COCs were isolated from large Graafian follicles and cultured in the presence of 33 and 50 Hz fields for 17–18 h. As shown in Table IGo, more than 80% of exposed and control COCs underwent normal cumulus mucification and similarly high percentages of oocytes reached the MII stage, as evidenced by first polar body extrusion. Assessment of oocyte chromatin configuration by Hoechst 33342 staining confirmed the presence of a normal MII configuration in all the experimental conditions tested (data not shown).


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Table I. Effects of 33 and 50 Hz field exposure on cumulus–oocyte complex (COC) mucification and oocyte meiotic maturation up to metaphase II (MII)
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In mammals, the production of a mature egg results from a coordinated sequence of events in the ovarian follicle involving various cell types, such as the oocyte and the surrounding granulosa–thecal cells. In the current study, by using a culture system which permits complete pre-antral follicle growth and differentiation (Boland et al., 1993Go), it was demonstrated that ELF–EMF exposure significantly affects the differentiation process of mouse follicles by diminishing the proportion of pre-antral follicles capable of complete growth and/or of developing antral cavities.

Both frequencies tested affected follicle development, though to a different extent. In fact, the 33 Hz-exposure more efficiently reduced antrum formation than the 50 Hz-exposure. The apparent lack of effect of the 50 Hz frequency on follicular growth can be, at least in part, explained by the fact that the values obtained in the experiments on follicular growth were derived from the measurement of all cultured follicle diameters, regardless of antrum formation. When other parameters of follicle development were related to antrum formation, a reduction in cell proliferation and 17ß-oestradiol production in the follicles with impaired antrum formation was observed. Conversely, the few follicles whose antrum formation was not affected by field exposure showed values similar to those obtained in the control follicles that developed antra.

The data presented suggest that ELF–EMF exposure has a detrimental effect on the physiological parameters of the majority of exposed follicles and that this detrimental effect on the somatic cells, in turn, determines an impaired ability to sustain normal oocyte differentiation. In fact, even though ELF–EMF exposure does not interfere with oocyte growth, a high percentage of the oocytes derived from exposed follicles without antrum do not complete nuclear maturation and remain arrested at the GV stage. This effect does not appear to affect the mechanism(s) controlling meiotic maturation per se since a high percentage of oocytes obtained from in-vivo-differentiated pre-ovulatory follicles progress up to MII stage despite ELF–EMF exposure.

Since a significant decrease in oestradiol production and granulosa cell proliferation has been related to granulosa cell apoptosis (Kaipia and Hsueh, 1997Go; Drummond and Findlay, 1999Go), an intriguing explanation for the current results may be that ELF–EMF is capable of inducing granulosa cell apoptosis. Indeed, data reported in the literature show that ELF–EMF exposure affects this process in various somatic cell types (Flipo et al., 1998Go; Ismael et al., 1998Go; Fanelli et al., 1999Go). However, exposure to ELF–EMF does not induce granulosa cell apoptosis despite the low oestradiol concentration and proliferation recorded, nor does it prevent this process in follicles cultured in the absence of serum to induce spontaneous cell death (McGee et al., 1997Go).

The potential mechanisms through which ELF–EMF promote these effects are not yet known. In other mammalian cell systems, weak fields interact with cells by altering free calcium concentrations, membrane-dependent signal transduction pathways as well as activities of key protein kinases (Goodman et al., 1995Go; Holian et al., 1996Go; Jahreis et al., 1998Go; Kristupaitis et al., 1998Go; Loschinger et al., 1998Go; Campbell-Beachler et al., 1998Go; Tuinstra et al., 1998Go). Thus, it may be postulated that ELF–EMF alter the follicle proliferative/differentiative programme by negatively affecting an as yet undefined regulatory mechanism in ovarian somatic cells. This possibility is further sustained by the fact that theca and granulosa cells play a central role in enhancing follicle development (McGee et al., 1997Go) by modulating the action of gonadotrophic hormones and the production of auocrine/paracrine factors (Spears et al., 1998Go).

In conclusion, besides the puzzling question regarding the impaired physiological processes, the possible negative role exerted by ELF–EMF on the reproductive potential of women chronically exposed to such fields is a problem that needs to be addressed.


    Acknowledgments
 
We thank Dr Raffaella Parroni for technical help; the National Hormone and Pituitary Distribution Program, NIDDKD, NIH for providing ovine FSH and Mr Lewis Baker for reviewing the English of the manuscript. This work was funded by grants from MURST to S.C., R.C. and M.G.C.


    Notes
 
4 To whom correspondence should be addressed at: Department of Biomedical Sciences and Technologies, University of L'Aquila, 67100 L'Aquila, Italy. E-mail: cecconi{at}univaq.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 23, 2000; accepted on July 18, 2000.