Effects of Hoechst 33342 staining and ultraviolet irradiation on the developmental competence of in vitro-matured porcine oocytes.

Hoechst 33342 (H342) in combination with ultraviolet (UV) irradiation is frequently used to assist the enucleation of porcine oocytes in somatic cell nuclear transfer programs. This work evaluated the effects of H342 (5 µg/mL for 12 min) staining and/or exposure to UV irradiation on fertilisability and developmental capacity of porcine oocytes matured in vitro. In Experiment 1, a total of 1388 mature oocytes were distributed in the following groups: Group 1: oocytes without treatment (Control), Group 2: oocytes stained with H342, Group 3: oocytes stained with H342 and UV irradiated for 30 sec, and Group 4: oocytes UV irradiated for 30 sec. Oocytes from each group were exposed to thawed spermatozoa and cultured for 18 h to assess fertilization parameters or for 7 d to evaluate embryo development. Sperm penetration (P < 0.001) and monospermy (P < 0.04) were lower in oocytes exposed to H342/UV (80.7 ± 4.5% and 30.7 ± 5.4%, respectively) than in oocytes from the control group (94.9 ± 4.3 and 50.0 ± 4.9, respectively). The oocytes exposed to H342/UV showed lower (P < 0.001) cleavage (49.8 ± 2.9%) and blastocyst (7.7 ± 2.9%) rates than oocytes from the other groups (range: 73.8 ± 2.9% to 77.7 ± 2.9% and 22.3 ± 2.9% to 30.9 ± 3.0%, respectively). Experiment 2 was designed to evaluate the effect of shorter UV irradiation (5 sec). A total of 1835 mature oocytes were separated into the same groups as those of Experiment 1. The fertilization parameters and the cleavage rates were not influenced by the different treatments. However, the oocytes exposed to H342 and UV irradiation for 5 sec showed a lower (P < 0.02) rate of blastocyst formation (15.2 ± 4.5%) than the oocytes from other groups (range: 26.1 ± 4.5% to 30.7 ± 4.5%). In conclusion, our results demonstrate that the combination of H342 staining with UV irradiation has a clear deleterious effect on the developmental ability of oocytes, with the effects being more intense with increased exposure to UV irradiation.

The effect of pulsed 900-MHz GSM mobile phone radiation on the acrosome reaction, head morphometry and zona binding of human spermatozoa.

Several recent studies have indicated that radiofrequency electromagnetic fields (RF-EMF) have an adverse effect on human sperm quality, which could translate into an effect on fertilization potential. This study evaluated the effect of RF-EMF on sperm-specific characteristics to assess the fertilizing competence of sperm. Highly motile human spermatozoa were exposed for 1 h to 900-MHz mobile phone radiation at a specific absorption rate of 2.0 W/kg and examined at various times after exposure. The acrosome reaction was evaluated using flow cytometry. The radiation did not affect sperm propensity for the acrosome reaction. Morphometric parameters were assessed using computer-assisted sperm analysis. Significant reduction in sperm head area (9.2 ± 0.7 µm² vs. 18.8 ± 1.4 µm²) and acrosome percentage of the head area (21.5 ± 4% vs. 35.5 ± 11.4%) was reported among exposed sperm compared with unexposed controls. Sperm-zona binding was assessed directly after exposure using the hemizona assay. The mean number of zona-bound sperm of the test hemizona and controls was 22.8 ± 12.4 and 31.8 ± 12.8 (p < 0.05), respectively. This study concludes that although RF-EMF exposure did not adversely affect the acrosome reaction, it had a significant effect on sperm morphometry. In addition, a significant decrease in sperm binding to the hemizona was observed. These results could indicate a significant effect of RF-EMF on sperm fertilization potential.

Light irradiation of mouse spermatozoa: stimulation of in vitro fertilization and calcium signals.

Irradiation of mouse spermatozoa by 630 nm He-Ne laser was found to enhance the intracellular calcium levels and fertilizing potential of these cells. The effect of light on calcium transport and on fertilization rate was abrogated in the absence of Ca2+ during the irradiation time, indicating that the effect of light is Ca2+ dependent. The stimulatory effect of light on Ca2+ uptake was abolished in the presence of a voltage-dependent Ca(2+)-channel inhibitor nifedipine, indicating the involvement of a plasma membrane voltage-dependent Ca2+ channel. Furthermore, the stimulatory effect of light was completely inhibited by the mitochondrial uncoupler FCCP, indicating that laser irradiation might affect the mitochondrial Ca2+ transport mechanisms. A causal association between laser irradiation, reactive oxygen species (ROS) generation and sperm function was indicated by studies with ROS scavengers, superoxide dismutase (SOD) and catalase, and exogenous hydrogen peroxide. The SOD treatment, which enhanced H2O2 production, resulted in increased Ca2+ uptake and enhanced fertilization rate. On the other hand, catalase, which decomposes H2O2, impaired the light-induced stimulation in Ca2+ uptake and the fertilization rate. Taken together, the data suggest that H2O2 might be involved in the irradiation effects, and indeed laser irradiation enhances the production of H2O2 by spermatozoa. These results indicate that the effect of 630 nm He-Ne laser irradiation is mediated through the generation of H2O2 by the spermatozoa and that this effect plays a significant role in the augmentation of the sperm cells' capability to fertilize metaphase II-arrested eggs in vitro.

Difference in types of radiation-induced structural chromosome aberrations and their incidences between Chinese and Syrian hamster spermatozoa.

The effects of ionizing radiations on sperm chromosomes were studied in the Chinese hamster (Crisetulus griseus) and the Syrian (golden) hamster (Mesocrisetus auratus). Testes of mature male Chinese hamsters (CH) were irradiated with X-rays (0.91, 1.82 and 3.63 Gy) and gamma-rays (1.10, 2.15, 2.95 and 4.01 Gy) at a single acute dosage, whereas the irradiation was done with lower doses of X-rays (0.45, 0.91 and 1.82 Gy) and gamma-rays (0.49, 0.99 and 1.98 Gy) in mature male Syrian hamsters (SH), taking the higher radiosensitivity of this species into consideration. They were mated with normal females within 6 days of exposure. Sperm-derived chromosomes were analyzed in 1125 and 1966 fertilized ova of the CH and the SH, respectively. In both species, there was no great difference in the induction of structural chromosome aberrations between X-irradiated and gamma-irradiated spermatozoa. Chromosome-type aberrations were predominantly induced. The incidence of breakage-type aberrations increased linearly, and that of exchange-type aberrations linear-quadratically with increase of dosage. A species-specific difference in chromosomal radiosensitivity of spermatozoa was clear. In spite of the same radiation dosage, the incidence of chromosomally abnormal spermatozoa in the SH was about twice as high as that in the CH (e.g. 27.0% vs. 14.7% at 0.91 Gy of X-rays). The incidences of breakage-type aberrations (69-89%) were far higher than those of exchange-type aberrations (11-31%) in the SH, while the disparity of the two incidences was much smaller in the CH (46-65% vs. 35-54%). Exchange-type aberrations consisted of both chromosome-type and chromatid-type in the SH, while almost all of them were of the chromosome-type in the CH. These results suggest that the DNA-repairing capacity of oocytes is much higher in the CH than in the SH. Moreover, it seems likely that radiation-induced sperm DNA damage is repaired with both pre-replication repair (excision repair) and post-replication repair systems in SH oocytes, whereas the excision repair system operate most exclusively in CH oocytes.

Induction of micronuclei in human sperm-hamster egg hybrids at the two-cell stage after in vitro gamma-irradiation of human spermatozoa.

The efficiency of the micronucleus test to assess radiation-induced chromosomal damage in human spermatozoa has been investigated. Micronuclei were scored in human sperm-hamster egg hybrids at the two-cell stage, after exposure of human spermatozoa to in vitro gamma-rays at doses of 0.00, 0.10, 0.25, 0.50, 1.00, 2.00, and 4.00 Gy. The relationship between the yield of micronuclei per two-cell stage as well as the percentage of two-cell stages with micronuclei and the different doses of irradiation were fitted to linear equations. To evaluate whether scoring micronuclei is useful for the quantification of chromosomal damage occurring in human spermatozoa, induced micronuclei at the different doses of sperm irradiation were compared to the induction of breaks and fragments in sperm-derived chromosomes. After interspecific fertilization of zona-free hamster oocytes by irradiated spermatozoa, a total of 699 fertilized eggs at the two-cell stage and a total of 387 sperm-derived complements were analyzed. The incidence of fertilized eggs with micronuclei at the two-cell stage coincided well with the incidence of sperm-derived chromosome breaks and fragments (e.g., 8.9% vs. 6.7% in the 0.25 Gy group and 52.8% vs. 58.6% in the 4.00 Gy group). A similar correlation was found between the number of micronuclei per two-cell stage and the number of breaks and fragments per sperm complement (0.09 vs. 0.07 in the 0.25 Gy group and 0.71 vs. 0.81 in the 4.00 Gy group). The results show that this test system can be used for the quantification of spontaneous or induced chromosomal damage in human spermatozoa.

Estimation of the quality of irradiated spermatozoa from cockerels using the classical method and zona-free hamster ova.

1. The quality of gamma irradiated spermatozoa from cockerels was investigated by measuring motility, oxygen consumption and ability to penetrate zona-free hamster ova. 2. Doses of 750 Gy slightly reduced motility and oxygen consumption; however, irradiated spermatozoa penetrated on average 56% (minimum 33%) of hamster ova. 3. The zona-free hamster ova test may be useful for estimating avian sperm quality.

An increased frequency of human sperm chromosomal abnormalities after radiotherapy.

13 cancer patients were studied before radiotherapy (RT) and at regular intervals after RT to determine the effect of RT on chromosomal abnormalities in sperm. The men were 19-47 years old and received testicular radiation doses of 0.4-5.0 Gray. Human pronuclear sperm chromosomes were analysed after penetration of zona-pellucida-free hamster eggs. Unfortunately the hamster egg penetration rates were exceedingly low, both before and after RT and this limited the number of sperm chromosome complements which could be analysed. Before RT, the frequency of abnormal sperm chromosome complements was 0% (0/9). After RT, the majority of men were azoospermic for 24 months but complements could be analysed from 4 men. In the first 12 months the frequency of abnormalities was 13% (1/8) and at 24 months it was 13% (7/55). By 36 months after RT, most men had recovered sperm production and the frequency of abnormalities in 8 men was 21% (18/86), which is significantly higher than the rate in control donors (8.5%). For individual men the range was 6-67%, and there was a significant correlation between testicular radiation dose and the frequency of sperm chromosomal abnormalities. The frequencies of both numerical and structural abnormalities were significantly increased after RT. This is the first evidence that radiation may increase the frequency of chromosomal abnormalities in human gametes.

Effect of gamma-radiation on fowl sperm function in vitro and in vivo.

Doses of up to 300 Gy of ionizing radiation had little effect on fowl sperm morphology, ATP content and motility when measured in vitro. Fertility of eggs from hens inseminated with spermatozoa receiving 50 Gy, in terms of post-oviducal development, was less than 4%. However, 35% appeared 'fertile' by macroscopic examination of the germinal disc of unincubated eggs. These contained few embryonic cells, although the vitelline membrane contained many trapped spermatozoa. After doses of 100 Gy or more, inseminated spermatozoa were not found in the vitelline membrane and no fertile or apparently fertile eggs were produced; nor did such spermatozoa enter the utero-vaginal sperm-host glands. Genetic transformation using fowl spermatozoa irradiated with doses in excess of 100 Gy appears to be an unlikely prospect.

A prospective serial study of the effects of radiotherapy on semen parameters, and hamster egg penetration rates.

Cancer patients were studied before radiotherapy (RT) and at regular intervals after treatment (1, 3, 12, 24, 36, 48 months) to determine the effect of radiotherapy on semen parameters and sperm function as assessed by the hamster egg penetration assay. The cancer patients received testicular radiation doses of 0.4 to 5.0 grays (40 to 500 rads). The pre-radiotherapy semen profile varied considerably but in general the profile was poor: 7/11 men had a sperm concentration less than 20 X 10(6)/ml and a total count of less than 50 X 10(6), while the hamster egg penetration rates were also very low with a mean of 5% (range 0% to 15%). This is the first study demonstrating that sperm function as well as sperm concentration is impaired in cancer patients pre-radiotherapy. At 3 and 12 months post-radiotherapy, 8/11 men were azoospermic. By 24 months 8/11 were producing sperm although only 2 had hamster egg penetration rates greater than 15%. All men studied at 36 months (4) and 48 months (3) post-radiotherapy had recovered spermatogenesis but hamster egg penetration rates were still poor. There was a highly significant inverse correlation between testicular radiation dose and subsequent sperm concentration and hamster egg penetration rates.