Interaction of microwave radiation with Turkey sperm

Summary Turkey sperm were exposed to 2.45 GHz microwave radiation in a temperature controlled waveguide apparatus. Temperature was maintained at 40.0 ± 0.5° C. The sperm were exposed for 30 min to a specific absorption rate (SAR) of 1, 10, or 50 mW/g. Before and following microwave exposure the following parameters were examined: percentage viability, percentage abnormal sperm, and release of the enzymes lactate hydrogenase (LDH) and glutamic oxalic transaminase (GOT). These parameters were not altered significantly by microwave exposure under the conditions tested.     

Penetration of mouse eggs at various intervals after insemination as influenced by concentration of sperm and strain of male

This study was conducted to determine the extent to which the percentage of mouse eggs that were penetrated by sperm at the end of the period of sperm penetration was due to the proportion of eggs penetrated per unit of time and to the span of time of sperm penetration. Female mice of ICR strain were inseminated 1.5 hr after ovulation with 5 X 10(6) sperm/50 microliter from males of DBA/2N, CF1 or C57BL/6N strains to determine the effect of the male. To determine the effect of concentration of sperm ICR females were inseminated with 2, 4, 6, or 8 X 10(6) sperm/50 microliter from CF1 males. Females were killed at various intervals after insemination and the eggs were recovered and examined for evidence of penetration by a sperm. The time intervals from both insemination to the onset of egg penetration and from insemination to cessation of penetration were similar for the three strains of males. Throughout the period of penetration of eggs a constant percentage of eggs was penetrated per hour for a particular strain of male. The relative percentage penetrated per hour very closely approximated the relative percentage of eggs finally penetrated for each strain of male. The percentage of eggs penetrated per hour was linearly positively related to the concentration of sperm inseminated. The final percentage of eggs penetrated depended primarily on the rate at which the eggs were penetrated during the period of sperm penetration and not on the length of the period of egg penetration which was constant.     

Thermal and metabolic responsiveness of Japanese quail embryos following periodic exposure to 2,450 MHz microwaves

Two studies were performed to determine if repeated exposure of the avian egg to microwaves can alter metabolism, temperature, and growth rate of embryos. Another aim was to supplement conventional heating with microwave heating and provide an optimal temperature for growth. Japanese quail (Coturnix coturnix japonica) eggs were exposed from day 1 through 15 of incubation (8 h/day) to sham or microwave (2,450 MHz) irradiation. Microwave exposures were at two power densities, 5 or 20 mW/cm2, and at three ambient temperatures (Tas), 30.0, 33.1, or 35.4 degrees C. Specific absorption rates for unincubated and 15-day-old incubated eggs were, respectively, 0.76 and 0.66 W kg-1 mW-1 cm-2 (i.e., 3.8 and 3.3 W/kg at 5 mW/cm2 and 15.2 and 13.2 W/kg at 20 mW/cm2). Eggs were concurrently sham exposed at each of five Tas, ranging from 27.9 to 37.5 degrees C. Tests were conducted during the 16th day of incubation (i.e., 1 day post-treatment), in the absence of microwaves, to determine metabolic rate of embryos and internal and external egg temperatures at different Tas. Repeated exposures to microwaves at 5 and 20 mW/cm2 at the same Ta (30 degrees C) increased wet-embryo mass on the 16th day by an average, respectively, of 9% and 61% when compared with predicted masses for embryos exposed at the same Ta in the absence of microwave radiation. There was no reliable indication, from post-treatment tests and comparisons with control embryos of similar mass, that repeated exposure to microwave radiation resulted in abnormal physiological development. Microwave radiation can be used to increase egg temperature and embryonic growth rate at Tas below normal incubation level without altering basic metabolic and thermal characteristics of the developing bird.     

In vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs.

In vitro fertilization of rat and mouse eggs by ejaculated or epididymal spermatozoa in chemically defined media was studied. Penetration rates by ejaculated sperm was very low (0 to 8%) in the rat, but 11 to 41% of eggs were penetrated by ejaculated sperm in the mouse. The optimal concentration of sperm for in vitro fertilization appears to be similar whether ejaculated or epididymal sperm were used. The time of sperm penetration in the mouse eggs, however, was delayed for one-half to one hour when ejaculated sperm were used. The importance of sodium pyruvate, sodium lactate and glucose in the medium containing bovine serum albumin for in vitro fertilization of rat eggs was examined. When rat eggs in cumulus clot were exposed to epididymal sperm preincubated for five hours, the presence of sodium pyruvate, sodium lactate and glucose was found to play an important role. When exposed to non-incubated epididymal sperm sodium pyruvate could be omitted without much decline of the fertilization rate. When the denuded eggs were exposed to non-incubated sperm, penetration rates were very low (0 and 5%) in the absence of pyruvate. It appears that although lactate, pyruvate and glucose are all important for in vitro fertilization of rat eggs, pyruvate can be supplied by the follicular cells surrounding the eggs.     

Serum enzymes in hemorrhaged Japanese quail after microwave irradiation during embryogeny

1. Japanese quail eggs were exposed to 2.45 GHz continuous wave microwave radiation at an incident power density of 5 mW/cm2 and a specific absorption rate of 4.03 mW/g during the first 12 days of embryogeny. 2. After hatching, serum biochemical changes in response to hemorrhagic stress were measured following a hemorrhage of 30% of the calculated total blood volume. 3. Lactate dehydrogenase, beta-glucuronidase, acid phosphatase, glucose and protein were not affected by microwave irradiation during embryogeny either before or after hemorrhage. 4. Microwave irradiation in ovo affected the response of serum glutamic oxaloacetic transaminase activity to hemorrhagic stress in Japanese quail.     

Mathematical simulation of dose–effect relationships for fish eggs exposed chronically to ionizing radiation

A mathematical model which simulates the observed dose-effect relationships for fish eggs exposed to chronic irradiation is presented. The model assumes that the exposed fish eggs may exist in one of the following states: normally developing, reversibly damaged, and lethally damaged. Reversible damages may be recovered by repairing mechanisms which are spent for the repairing processes. The model was applied to describe the observed differences in effects of chronic exposure for quickly (2 weeks) and slowly (up to 20 weeks) developing fish eggs. Calculations were performed for dose rates of chronic irradiation ranging from 10 to 300 mGy/day. Two types of radiation effects were considered—the effect on eggs survival (percentage of survived eggs at time t), and the depletion of the repairing pool (in percentage of its maximal value). The model predictions have been compared with the experimental data from the EPIC database. This comparison showed that the model adequately describes the radiation effects in fish eggs of different species, within a wide range of chronic radiation exposures.     

Dominant lethal studies in male mice after exposure to 2.45 GHz microwave radiation

Adult male mice had the lower halves of their bodies exposed in a waveguide system to 2.45 GHz microwave radiation for 30 min. The half body dose-rate of 43 W kg-1 had been shown in a previous study [7] to deplete severely the heat-sensitive stages of sperm production. The males were mated at intervals to adult hybrid females over the following 8-10 weeks. There was no significant reduction in post-implantation survival, suggesting that the microwave exposure did not have a mutagenic effect on the male germ cells. However, pregnancy rate was significantly reduced in weeks 3, 4, 5 and 6; reaching a minimum of about 10% of the control value in weeks 4 and 5. The occurrence of low values in weeks 4 and 5 correlated well with the expected reductions in sperm count due to the pattern of depletion of the spermatogenic epithelium of the testes. Thus it was concluded that the reduced pregnancy rate resulted from reduced male fertility. Pre-implantation survival can also be affected by reduced sperm count [8] and was significantly reduced in this study but it correlated less well with the anticipated heat response. A further study is in progress looking at the contribution of sperm count and sperm abnormality to the results.     

The effect of the acrosome reaction on the respiratory activity and fertilizing capacity of echinoid sperm.

We have examined the relationship between the acrosome reaction, sperm respiration, and fertilization using gametes of the sea urchin Strongylocentrotus purpuratus. The results indicate that when sperm are exposed to jelly coat isolated from homologous eggs, the following sequence of events occurs: (1) Sperm undergo the acrosome reaction within 30 sec with little or no loss in their capacity to fertilize eggs; (2) by 60 sec there is a dramatic decrease in fertilizing capacity which stabilizes after 4 or 5 min at a greatly reduced level; (3) by 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration becomes detectable and continues for 8 to 10 min, finally stabilizing at a greatly reduced rate. This decrease in respiration rate is paralleled by a decline in sperm motility. The effects of jelly coat on the acrosome reaction, sperm respiration, and motility are species specific. From these results we conclude that sperm which have undergone the acrosome reaction retain full fertilizing capacity for a very short time. The rapid decline in fertilizing capacity is followed by a decrease in respiration rate and motility.     

Leukocyte numbers in hemorrhaged Japanese quail after microwave irradiation in ovo

1. Coturnix coturnix japonica eggs were exposed to 2.45-GHz continuous wave microwave radiation at an incident power density of 5 mW/cm2 (and a specific rate of 4 mW/g) during the first 12 days of embryogeny. After hatching, hematological changes in response to an acute hemorrhage were measured in exposed and nonexposed (control) juveniles of both sexes. 2. Exposure did not affect erythroid cell numbers either before or after hemorrhage. 3. Exposure affected the recovery of lymphocyte and heterophil numbers after hemorrhage, but the effect was sex-limited. 4. These data indicate that microwave irradiation during embryogeny in ovo affects the ability of Japanese quail to recover from an acute and voluminous hemorrhage and that these radiation effects are sex-limited and consistent with a previous report.     

Influences of DMP on the fertilization process and subsequent embryogenesis of abalone (Haliotis diversicolor supertexta) by gametes exposure

Di-methyl phthalate (DMP), a typical endocrine disrupting chemical (EDC), is ubiquitously distributed in aquatic environments; yet studies regarding its impact on gametes and the resulting effects on embryogenesis in marine gastropods are relatively scarce. In this study, the influences of DMP on the gametes and subsequent developmental process of abalone (Haliotis diversicolor supertexta, a representative marine benthic gastropod) were assessed. Newborn abalone eggs and sperm were exposed separately to different DMP concentrations (1, 10 or 100 ppb) for 60 min. At the end-point of exposure, the DMP-treated eggs and sperm were collected for analysis of their ultra-structures, ATPase activities and total lipid levels, and the fertilized gametes (embryos) were collected to monitor related reproductive parameters (fertilization rate, abnormal development rate and hatching success rate). Treatment with DMP did not significantly alter the structure or total lipid content of eggs at any of the doses tested. Hatching failures and morphological abnormalities were only observed with the highest dose of DMP (100 ppb). However, DMP exposure did suppress sperm ATPase activities and affect the morphological character of their mitochondria. DMP-treated sperm exhibited dose-dependent decreases in fertilization efficiency, morphogenesis and hatchability. Relatively obvious toxicological effects were observed when both sperm and eggs were exposed to DMP. Furthermore, RT-PCR results indicate that treatment of gametes with DMP changed the expression patterns of physiologically-regulated genes (cyp3a, 17β-HSD-11 and 17β-HSD-12) in subsequent embryogenesis. Taken together, this study proofed that pre-fertilization exposure of abalone eggs, sperm or both to DMP adversely affects the fertilization process and subsequent embryogenesis.     

The production of cloned fish in the medaka (Oryzias latipes)

The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41 degrees C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85-95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41 degrees C for 2 min at 2-3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85-95 min after insemination arrests the first cleavage, while heat shock at 2-3 min after insemination arrests the second meiotic division. Medaka clones have been produced by the following method: Eggs from orange-red or variegated variety were activated by UV-irradiated, genetically impotent sperm of wild-type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone-containing diet (40 micrograms/gm diet) to produce sex-reversed males, which were mated with brood females, and thus two cloned lines were obtained.     

Mobile phone usage and male infertility in Wistar rats

A significant decrease in protein kinase C and total sperm count along with increased apoptosis were observed in male Wistar rats exposed to mobile phone frequencies (2 h/day x 35 days at 0.9 W/kg specific absorption rate). The results suggest that a reduction in protein kinase activity may be related to overproduction of reactive oxygen species (ROS) under microwave field exposure. Decrease in sperm count and an increase in apoptosis may be causative factor due to mobile radiation exposure leading to infertility.     

THE FERTILIZING CAPACITY OF SEA URCHIN SPERM RAPIDLY DECREASES AFTER INDUCTION OF THE ACROSOME REACTION*

When immotile, flagella-less sperm were added to acid-dejellied eggs of Strongylocentrotus purpuratus 11% of the eggs fertilized. Addition of soluble egg jelly increased the percentage fertilization to 90.5. Over 50% of the sperm exposed to egg jelly had undergone the acrosome reaction compared to only 3–5% in the absence of jelly. Egg jelly was added to flagella-less sperm to induce the acrosome reaction and dejellied eggs added at various times thereafter. The fertilizing capacity of the sperm decreased with first order kinetics with 50% loss by 23 sec after induction of the acrosome reaction. Intact, motile sperm bind to formaldehyde-fixed eggs with maximum binding occurring 40 sec after sperm addition. After 40 sec the sperm begin to detach from the fixed eggs and by 240 sec none remain attached. Sperm detachment from fixed eggs and loss of fertilizing capacity after the acrosome reaction show a close temporal correlation.     

Comparison of heterospermic and homospermic inseminations as measures of male fertility

This study attempted to determine a basis for the previously observed greater sensitivity of heterospermic tests when compared to homospermic tests for detecting differences in fertility between males. In theory, the results of heterospermic tests are an indication of the proportion of eggs fertilized per unit time whereas results of homospermic inseminations measure only the cumulative or final proportion of eggs fertilized. The fertilizing ability of sperm from males of CF1 and C57BL/6N strains of mice was compared homospermically using both relatively high and low concentrations of sperm and by measuring the proportion of eggs penetrated per unit of time. The fertilizing ability of sperm from these strains was also compared using heterospermic inseminations. When females were inseminated with a high concentration of sperm, males of both strains fertilized a high and indistinguishable percentage of eggs when examined after 30 hr. When females were inseminated with either a low concentration of sperm or when the proportion of eggs penetrated was measured at 5 hr, differences between strains of mice were distinguishable. Heterospermic insemination further magnified the observed difference between strains. The results of this study confirm that measuring the percentage of eggs fertilized per unit of time can enhance the magnitude of differences between males in fertility as compared to measuring only the final percentage of eggs fertilized. Measuring the percentage of eggs fertilized per unit of time does not, however, entirely account for the large differences observed between fertility of males when they are compared using heterospermic inseminations.     

Lipid peroxidation was associated to the impairment of the fertilizing capability of gilthead sperm exposed to surfactants

The present study was designed to determine whether lipid peroxidation was associated with the impairment of the fertilizing capability of gilthead sperm after acute exposure to anionic surfactant Sodium Dodecyl Sulphate (SDS). Spawned eggs and sperm were collected from adult giltheads. Sperm suspensions (100,000,000 spermatozoa/mL) were dosed separately with different concentrations of SDS (0.6, 1.5, 3 and 6 mg/L) for 60 minutes. After this period, sperm samples were randomly distributed for both outcome measurements: fertilization percentage or lipid peroxidation assessment. On one hand, exposed sperm and unexposed eggs were combined for 20 minutes during which fertilization took place. Fertilization, defined as the presence of a fertilization envelope, was assessed by microscopic observation. On the other hand lipid peroxidation on exposed gilthead sperm was determined by estimating the production of malondialdehyde (MDA). Acute exposure to SDS caused a significant inhibitory effect on fertilization success in gilthead. It also increased significantly lipid peroxidation in exposed sperm. Furthermore, a strong but negative statistical association was found between fertilizing capability and lipid peroxidation gilthead sperm exposed to SDS. Although extrapolation from the laboratory to the field requires caution, the results of this work demonstrated that the impairment of fertilization was significantly associated with lipid peroxidation induced by acute exposure to SDS. Consequently lipid peroxidation may be recommended as an early-warning bioindicator of exposure to surfactants. Further studies are required.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: I. A fertility assay for fresh semen

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona-free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within-liposome-concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of ?.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was ?.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona-free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.     

Response of Japanese quail to hemorrhagic stress after exposure to microwave radiation during embryogeny

Coturnix coturnix japonica eggs were exposed to 2.45 GHz continuous wave microwave radiation at an incident power density of 5 mW/cm2 (SAR = 4 mW/g) during the first 12 days of embryogeny. After hatching, hematologic changes in response to an acute hemorrhage were measured in exposed and nonexposed (control) juveniles and adults of both sexes. Reticulocyte numbers and percentages were depressed below control numbers at 24 hr postphlebotomy in exposed adult females. Lymphocyte numbers were depressed below control levels at 24 hr postphlebotomy in exposed juvenile and adult males. At 72 hr heterophil numbers were depressed in exposed juvenile and adult males. These data suggest that microwave irradiation during embryogeny affects the ability of Japanese quail to recover from an acute and voluminous hemorrhage and that these radiation effects are small.     

Stress proteins are not induced in mammalian cells exposed to radiofrequency or microwave radiation

The induction of stress proteins in HeLa and CHO cells was investigated following a 2 h exposure to radiofrequency (RF) or microwave radiation. Cells were exposed or sham exposed in vitro under isothermal (37 ± 0.2 °C) conditions. HeLa cells were exposed to 27- or 2450 MHz continuous wave (CW) radiation at a specific absorption rate (SAR) of 25 W/kg. CHO cells were exposed to CW 27 MHz radiation at a SAR of 100 W/kg. Parallel positive control studies included 2 h exposure of HeLa or CHO cells to 40 °C or to 45 μM cadmium sulfate. Stress protein induction was assayed 24 h after treatment by electrophoresis of whole-cell extracted protein labeled with [³⁵S]-methionine. Both cell types exhibited well-characterized responses to the positive control stresses. Under these exposure conditions, neither microwave nor RF radiation had a detectable effect on stress protein induction as determined by either comparison of RF-exposed cells with sham-exposed cells or comparison with heat-stressed or Cd⁺⁺ positive control cells. Bioelectromagnetics 18:499–505, 1997. © 1997 Wiley-Liss, Inc.     

Mouse gamete interactions: The zona pellucida is the site of the acrosome reaction leading to fertilization in vitro

The question of whether the acrosome reaction, which leads to fertilization, occurs in intact sperm bound to the zona pellucida of the egg or in intact sperm before contact with the egg, was addressed by assessing the effect of 3-quinuclidinyl benzilate (QNB) on the two types of acrosome reaction. QNB is a specific inhibitor of the fertilization of zona-intact mouse eggs by mouse sperm. Mouse spermatozoa in suspension underwent acrosome reactions at a low rate, which could be accelerated by addition of 5 μM divalent cation ionophore A23187; the occurrence of such acrosome reactions was not inhibited by QNB. The rate at which acrosome reactions occurred in sperm bound to the zona pellucida of cumulus-free eggs, bound to isolated zonae, or exposed to acid-solubilized zona components, was greatly accelerated relative to that observed in the absence of zonae. These acrosome reactions were strongly inhibited by QNB at concentrations which inhibit the fertilization of zona-intact mouse eggs in vitro. These data suggest that the zona pellucida can induce acrosome reactions in mouse spermatozoa and that these acrosome reactions are the ones which lead to the fertilization of zona-intact eggs. In contrast, the acrosome rection in sperm which are not in contact with the zona is not associated with fertilization of zona-intact eggs.