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
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Abstract |
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Key words: apoptosis/extremely low frequency electromagnetic field/meiotic maturation/pre-antral follicles
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Introduction |
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The effects of extremely low-frequency electromagnetic fields (ELFEMF; 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 ELFEMF on cell functions are somewhat controversial (McCann et al., 1998
) since ELFEMF 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., 1995
; Mevissen et al., 1995
). Indeed, the application of a wide range of field intensities (3100 Hz) can negatively affect haemopoiesis (Bonhomme-Faivre et al., 1998
; Mevissen et al., 1998
), enhance growth rates of transformed cells in some human epithelial cancers (Watson et al., 1998
), and even reduce the effect of antineoplastic drugs (Harland and Liburdy, 1997
). No genotoxic effects, in terms of DNA damage, have been reported for ELFEMF-exposed cells (Fiorani et al., 1992
; Morandi et al., 1996
; McCann et al., 1998
; Scarfi et al., 1999
) 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 hypoxanthineguanine phosphoribosyltransferase (HGPT) have been described for various cell types (Mather et al., 1990
; Miyakoshi et al., 1997
; Campbell-Beachler et al., 1998
; Han et al., 1998
; Jahreis et al., 1998
; Lagroye and Poncy, 1998
; Lin et al., 1998
; Loberg et al., 1999
). Various reports suggest that the interaction site for ELFEMF is the plasma membrane, since exposure determines altered Ca2+ influx (Conti et al., 1985
; Liburdy, 1992
; Flipo et al., 1998
; Fanelli et al., 1999
; Walleczek et al., 1999
), as well as the clustering of integral plasma membrane proteins (Bersani et al., 1997
). In addition, ELFEMF-induced oscillations of intracellular Ca2+ concentration (Loschinger et al., 1998
) have been correlated with changes in DNA, RNA and protein synthesis activities (Liburdy, 1992
). More recently, the application of ELFEMF has been involved in the reduction (Fanelli et al., 1999
) or stimulation (Flipo et al., 1998
; Ismael et al., 1998
) 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 ELFEMF 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, 1100 mT) can affect the proliferative/differentiative capacity of mouse spermatogonia (Furuya et al., 1998), but does not induce clastogenic effects on human sperm chromosomes (Tateno et al., 1998
). 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 ELFEMF on important aspects of mammalian oogenesis are available. As a consequence, this study was designed to evaluate whether ELFEMF 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.
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Materials and methods |
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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 ELFEMF exposure system
The ELFEMF 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 02.8 mT (028 G), with a square wave from 175 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 (), 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 (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 ELFEMF. 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 1618 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., 1991). 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 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., 1991). Granulosa cells obtained by puncturing ovaries of 22 day old mice were either cultured in DMEM supplemented with 0.1% BSA for 2448 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 celloocyte complex isolation and culture
Ovaries from PMSG-primed mice were excised and placed in a HEPES-buffered medium (Quinn et al., 1982). Cumulusoocyte 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 1618 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 TukeyKramer test for comparison of multiple groups.
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Results |
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Effect of ELFEMF exposure on follicle cell apoptosis
In order to determine whether ELFEMF 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 ELFEMF 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., 1997). The proportion of apoptotic bodies in granulosa cells obtained from freshly harvested (time = 0) and 2448 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 4
, 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 ELFEMF exposure (data not shown).
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Discussion |
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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 ELFEMF 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 ELFEMF 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 ELFEMF exposure.
Since a significant decrease in oestradiol production and granulosa cell proliferation has been related to granulosa cell apoptosis (Kaipia and Hsueh, 1997; Drummond and Findlay, 1999
), an intriguing explanation for the current results may be that ELFEMF is capable of inducing granulosa cell apoptosis. Indeed, data reported in the literature show that ELFEMF exposure affects this process in various somatic cell types (Flipo et al., 1998
; Ismael et al., 1998
; Fanelli et al., 1999
). However, exposure to ELFEMF 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., 1997
).
The potential mechanisms through which ELFEMF 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., 1995; Holian et al., 1996
; Jahreis et al., 1998
; Kristupaitis et al., 1998
; Loschinger et al., 1998
; Campbell-Beachler et al., 1998
; Tuinstra et al., 1998
). Thus, it may be postulated that ELFEMF 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., 1997
) by modulating the action of gonadotrophic hormones and the production of auocrine/paracrine factors (Spears et al., 1998
).
In conclusion, besides the puzzling question regarding the impaired physiological processes, the possible negative role exerted by ELFEMF on the reproductive potential of women chronically exposed to such fields is a problem that needs to be addressed.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 23, 2000; accepted on July 18, 2000.