Relative biological effectiveness (RBE) of 252Cf fission neutrons for the induction of chromosome damage in human spermatozoa.

Effects of 60Co gamma-rays and 252Cf neutrons on human sperm chromosomes were studied using our interspecific in vitro fertilization system to estimate relative biological effectiveness (RBE) of neutrons. Semen samples were exposed to 0.5, 1.0 and 2.0 Gy of 60Co gamma-rays at 1.7 cGy/ min and 0.25, 0.5 and 1.0 Gy of 252Cf radiation at 1.3-1.7 cGy/ min. In the 60Co experiment, 509 spermatozoa from controls and 902 spermatozoa from the irradiated groups were karyotyped, while in the 252Cf experiment 460 control and 804 irradiated spermatozoa were analysed. In both 60Co and 252Cf experiments, incidences of spermatozoa with radiation-induced structural chromosome aberrations increased linearly with increase of dosage. The RBE of 252Cf neutrons for the induction of chromosomally abnormal spermatozoa was estimated to be 1.6. The number of induced structural chromosome aberrations per spermatozoon also increased linearly. The RBE of neutrons for this index was 2.0. Among structural chromosome aberrations observed, chromosome-type breaks were predominant in both 60Co and 252Cf experiments, and they showed a significant linear dose-dependent increase. Other types of aberrations such as chromosome-type exchanges and chromatid-type breaks also increased linearly with increase in dose. The RBEs of 252Cf neutrons for the induction of these three types of aberrations were 1.6, 3.2 and 3.9, respectively. Thus, the RBEs of neutrons for the induction of chromosome aberrations were smaller in human spermatozoa than in human lymphocytes, and mouse spermatogonia and embryos. This result is discussed from the point of view of DNA-repairing capacity of oocytes.

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 aneuploidy in Chinese hamster oocytes following in vivo treatments with trimethoxybenzoic compounds and their analogues.

Many inhibitors of tubulin polymerization have a trimethoxybenzene ring in their molecules. Such trimethoxybenzoic compounds and their analogues may therefore have a potency to induce meiotic nondisjunction of oocytes. In this study, a single dose of reserpine (0.5 microgram/g body weight), podophyllotoxin (20.0 micrograms/g b.w.), trimethoxybenzoic acid (500.0 micrograms/g b.w.) or vinblastine sulfate (3.0 micrograms/g b.w.) was injected intraperitoneally to mature female Chinese hamsters at the onset of the first meiotic spindle formation of oocytes. Within 6 h after spontaneous ovulation, MII oocytes were collected from the oviducts for morphological examination and cytogenetic analysis. The incidence of morphologically abnormal oocytes with unusually large first polar body or bodies increased significantly after the treatment with reserpine (18/202; 8.9%), podophyllotoxin (28/172; 16.3%) and vinblastine sulfate (63/197; 32.0%), as compared with the control (3/214; 1.4%). Chromosome analysis of oocytes revealed that podophyllotoxin and vinblastine sulfate were effective in inducing aneuploidy (62/154; 40.3% and 128/156; 82.1% vs. 3/198; 1.5% of the control) by inhibiting the formation of spindle microtubules at the first meiosis. Aneuploids were found more frequently in morphologically abnormal oocytes than in normal oocytes. No aneugenic activity of reserpine and trimethoxybenzoic acid was observed. These results indicate that trimethoxybenzoic compounds do not necessarily exhibit aneugenic activity.

Chromosome analysis of human spermatozoa exposed to antineoplastic agents in vitro.

We studied in vitro the cytogenetic effects of six antineoplastic agents, bleomycin (BM), cyclophosphamide (CP), daunomycin (DM), methyl methanesulfonate (MMS), mitomycin C (MMC) and triethylenemelamine (TEM) on spermatozoa, using an interspecific in vitro fertilization system between zona-free hamster oocytes and human or bull spermatozoa. In preliminary experiments with bull spermatozoa, clastogenic effects were clearly shown with BM, DM, MMS and TEM, but not with CP and MMC. In main experiments, the effects of the first four chemicals were studied in detail with human spermatozoa. Total numbers of 585 and 512 spermatozoa were karyotyped in the control and the chemical-treated groups respectively. The incidence of spermatozoa with structural chromosome aberrations was 34.5%, 53.0%, 59.3%, and 55.6% in the BM (50 micrograms/ml, 90 min), DM (0.1 microgram/ml, 90 min), MMS (100 micrograms/ml, 120 min) and TEM (0.1 micrograms/ml, 120 min) groups respectively, each showing a significantly higher incidence than the matched controls (10.1-13.5%). Breakage-type aberrations were more frequent than exchange-type aberrations in the BM, MMS and TEM groups, while the exchange-type aberrations were more frequent in the DM group. Exchanges were mainly of the chromatid type in the DM, MMS and TEM groups, while chromosome-type exchanges occurred more frequently in the BM group. These results are discussed in relation to previous data on chemical-induced chromosome aberrations in mammalian somatic cells and in mouse spermatozoa.

Sperm chromosome study of two bulls heterozygous for different Robertsonian translocations.

Haploid chromosomes of a total of 354 spermatozoa from two bulls heterozygous for different Robertsonian translocations, a Holstein-Friesian bull carrying a t(1;21) and a Japanese Black bull carrying a t(7;21), were analyzed using an interspecific in vitro fertilization system with zona-free hamster oocytes. The proportion of chromosomally normal and balanced spermatozoa was approximately equal in both carriers (51.8% and 47.0% in the 1/21 carrier, and 47.3% and 50.0% in the 7/21 carrier). The combined incidences of normal and balanced spermatozoa, i.e., incidences of spermatozoa resulting from alternate meiotic segregation were very high (98.8% and 97.3%) in both carrier. On the contrary, the incidences of chromosomally unbalanced spermatozoa resulting from adjacent meiotic segregation were only 0.6% and 2.7%. These results indicate that the alternate segregation of a trivalent chromosome is predominant in these Robertsonian translocation carriers.

Chromosomal analysis of unfertilized human oocytes prepared by a gradual fixation-air drying method.

Two hundred and sixty-five unfertilized human metaphase II (M II) oocytes from an in vitro fertilization program were studied cytogenetically using our chromosomal technique, a gradual fixation-air drying method. Of the 265 oocytes, 185 (70%) were successfully karyotyped. There were 21 aneuploids (11.4%) consisting of 8 hyperhaploids (4.3%), 11 hypohaploids (5.9%) and 2 complex cases (1.1%). There were also 9 structural anomalies (4.9%) and 18 diploids (9.7%). In aneuploidy, the loss or gain of dyads (so-called nondisjunction) occurred more frequently than the loss or gain of monads (so-called predivision). The frequency of abnormally behaved chromosomes (segregation errors) due to nondisjunction, anaphase lag and predivision was studied among the seven chromosomal groups (A-G) and compared with the frequency expected from an equal probability of segregation errors in each of the 23 chromosomes. The observed frequency was somewhat higher than the expected frequency in groups E and G but the difference was not statistically significant in either group. These results were discussed in relation to previous studies on human M II oocyte chromosomes.

A freezing and thawing method of hamster oocytes designed for both the penetration test and chromosome assay of human spermatozoa.

Superovulated hamster oocytes were cryopreserved and thawed according to our carefully designed procedures. More than 90% (92 +/- 4%) of oocytes survived freezing and thawing. They were proven to be well conserved, showing excellent performance comparable to freshly ovulated oocytes in the human sperm penetration test (proportion of penetrated ova: 94.7% vs. 93.6%) and human sperm chromosome analysis (proportion of metaphasic ova: 81.8% vs. 83.6%). There was no statistically significant difference in the incidences of sperm chromosome aberrations between assays using fresh and frozen-thawed oocytes. In addition, there was no statistically significant increase of aberrations in female pronuclear (hamster) chromosomes. This freezing-thawing method was found to be reliable, yielding viable hamster oocytes of high quality.

Micronucleus test in 2-cell embryos as a simple assay system for human sperm chromosome aberrations.

A micronucleus test method to assess radiation-induced chromosomal damage in human spermatozoa is described, and its efficiency examined by comparison with that of sperm chromosome analysis. Human spermatozoa were exposed in vitro to 1.11 and 2.13 Gy of 137Cs gamma-rays at a dose rate of 1.36 Gy/min. After interspecific in vitro fertilization of irradiated spermatozoa with zona-free hamster oocytes, a total of 193 monospermic eggs were examined with the micronucleus test at the 2-cell stage, and a total of 304 male pronuclear chromosome plates were analyzed according to our established method. The incidence of 2-cell embryos with micronuclei coincided well with the incidence of spermatozoa with chromosomal breaks and fragments (51.6% vs. 50.3% in the 1.11-Gy group and 82.7% vs. 79.3% in the 2.13-Gy group). A similar correlation was also found between the number of micronuclei per embryo and the number of breaks and fragments per spermatozoon (0.85 vs. 0.88 and 1.50 vs. 1.45 in the 2 dose groups, respectively). These results indicate that our micronucleus test is useful as a simple and rapid method for assessing the clastogenic effects of various environmental mutagens on human sperm chromosomes.

A method for chromosome analysis of ram spermatozoa using zona-free hamster oocytes.

A method for displaying ram spermatozoan chromosomes using the interspecific zona-free hamster oocyte penetration was described to distinguish X- and Y-bearing spermatozoa. Semen samples from four rams were frozen and stored in liquid nitrogen. After thawing the samples, motile spermatozoa were collected by the swim-up method and treated with ionophore A23187 for the purpose of facilitating their capacitation. Slides were prepared by the gradual fixation-air dry method. The rates of oocyte penetration, first cleavage metaphase, and the number of ova that were karyotyped successfully were 67.9, 60.8 and 40.6%, respectively. The overall success rate (number of spermatozoa karyotyped/number of oocytes used for insemination) was 47.9%. A total of 1009 spermatozoa were analyzed, and the ratio of X- and Y-bearing spermatozoa was 508:501.

Dose-response relationship for the induction of structural chromosome aberrations in human spermatozoa after in vitro exposure to tritium beta-rays.

The effects of tritium (HTO) beta-rays on human sperm chromosomes were studied using our interspecific in vitro fertilization system between human spermatozoa and zona-free hamster oocytes. Semen samples were treated with media containing 1.53-24.3 mCi/ml HTO for about 80 min. 1290 spermatozoa from the controls and 1842 spermatozoa from the irradiated groups were karyotyped. The incidence of spermatozoa with structural chromosome aberrations increased linearly with increasing dosage. Breakage-type aberrations occurred far more frequently than exchange-type. Chromosome-type aberrations appeared far more frequently than chromatid-type. All of these types of aberrations showed linear dose-dependent increases. The RBE values of HTO beta-rays relative to X-rays were calculated for the above-mentioned 5 indices, respectively. Their RBE values ranged from 1.89 to 3.00 when the absorbed dose was estimated to be the minimum, whereas the values ranged between 1.04 and 1.65 when the absorbed dose was estimated to be the maximum.

Types of structural chromosome aberrations and their incidences in human spermatozoa X-irradiated in vitro.

We studied the effects of in vitro X-irradiation on human sperm chromosomes, using our interspecific in vitro fertilization system between human spermatozoa and zona-free hamster oocytes. 28 semen samples from 5 healthy men were exposed to 0.23, 0.45, 0.91 and 1.82 Gy of X-rays. Totals of 2098 and 2862 spermatozoa were karyotyped in the control and the irradiated groups, respectively. The incidence of spermatozoa with X-ray-induced structural chromosome aberrations (Y) increased linearly with increasing dosage (D), being best expressed by the equation, Y = 0.08 + 34.52 D. The incidence of breakage-type aberrations was more than 9 times higher than that of exchange-type aberrations. Both of them showed linear dose-dependent increases, which were expressed by the regression lines, Y = -0.014 + 0.478 D and Y = -0.010 + 0.057 D, respectively. The incidence of chromosome-type aberrations was about 6 times higher than that of chromatid-type aberrations. Their dose-dependent increases were expressed by the regression lines, Y = -0.015 + 0.462 D and Y = -0.006 + 0.079 D, respectively. These results are discussed in relation to the previous data obtained with gamma-rays. The repair mechanism of X-ray-induced sperm DNA lesions is also discussed.

Effects of neonatal ovarian X-irradiation in the Chinese hamster. II. Absence of chromosomal and developmental damages in surviving oocytes irradiated at the pachytene and resting dictyate stages.

Ovaries of Chinese hamsters were irradiated with 1 Gy of X-rays on day 0, 4, 8 and 14 after birth, and the late effects on pachytene and resting dictyate oocytes, which had been proven to be radioresistant and to survive acute oocyte-killing, were studied in the progeny of the irradiated females. Thirty hours after mating, fertilized ova were collected and female pronuclear chromosomes were karyotyped. On day 18.5 of gestation, the number of corpora lutea and implantation sites were counted and the live and dead fetuses were examined phenotypically. There was no increase of chromosome aberrations in female pronuclei of one-cell embryos of the irradiated groups. There was no significant difference in the incidences of pre- and postimplantation deaths and of congenital malformations between the irradiated groups and the non-irradiated controls. These findings indicated clearly that oocytes at the pachytene and resting dictyate stages which had survived the acute oocyte-killing effects of X-rays were also free from late deleterious effects on genetic and developmental abilities. The radiosensitivity of oocytes of the Chinese hamster was compared with that of the mouse with respect to chromosome aberrations, lethal mutations and congenital anomalies, and the difference between both species was discussed.

Effects of neonatal ovarian X-irradiation in the Chinese hamster. I. Correlation between the age of irradiation and the fertility span.

The fertility span between the first and the last estrus was compared among female Chinese hamsters exposed to 1 Gy X-rays on neonatal day 0, 2, 4, 6, 8, 10 and 14. The fertility span changed dramatically according to the age at the time of irradiation. The females irradiated on day 4, 6 and 8 showed a definite delay in sexual maturation and a very early cessation of the estrous cycles, while those irradiated on day 10, 2 and 14 (in this order) showed a much longer, nevertheless significantly shortened, fertility span. Reduction in the number of juvenile oocytes by acute killing was responsible for this shortened reproductive life. The present study indicated again that the sensitivity of the oocytes to the lethal effects of X-rays is unquestionably meiotic stage-dependent.

Cardiovascular sympathetic hypofunction in muscle contraction headache and migraine.

There have been some recent reports proposing that muscle contraction headache (MCH) and migraine are similar and may have a common etiology. It has been hypothesized that derangement of the autonomic nervous system (ANS) plays an important role in the pathogenesis of migraine. However, reports on the ANS function in MCH have rarely been submitted. Therefore, in this report, MCH patients were investigated as well as migraine patients. The cardiovascular reflex responses by orthostatic test, isometric work test and the pulse rate (R-R interval) variation in fifteen MCH patients and fifteen migraine patients were recorded during headache-free intervals. The plasma norepinephrine (NE) levels were also measured throughout the orthostatic tests. Fifteen healthy subjects served as the age-matched control group. In the MCH group and the migraine group, blood pressure immediately after going from the supine to the erect position decreased more significantly than in the control group. The basal NE level was significantly low in both the MCH group and the migraine group, in comparison with the control group. MCH patients as well as migraine patients showed cardiovascular sympathetic hypofunction.

A chromosomal method to distinguish between X- and Y-bearing spermatozoa of the bull in zona-free hamster ova.

The interspecific in-vitro fertilization system using zona-free hamster ova was adopted to distinguish chromosomally between X- and Y-bearing bull spermatozoa. Frozen semen samples were thawed and washed with modified BWW medium including 0.3% BSA (pH 7.2) to remove cryoprotective medium. Motile spermatozoa were recovered from the semen by the 'swim-up' method. These spermatozoa were treated with ionophore A23187 to facilitate capacitation. Adequate capacitation of spermatozoa was found by their movement patterns and the insemination was performed at the optimum time. The fertilized ova were cultured in Medium TC199 with 10% FBS including podophyllotoxin and vinblastine until they reached first cleavage metaphase. Chromosome slides were prepared by our gradual fixation-air drying method. The success rate of the method was 56% of the number corresponding to the zona-free ova used. Altogether 1116 spermatozoa from 4 different bulls were successfully analysed and the ratio of X- and Y-bearing spermatozoa was 542:574 (P greater than 0.3).

An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova.

We have developed an improved method for analyzing human sperm chromosome, using zona-free hamster ova. Our main improvements of methodology are as follows: (1) Fertilization rate of hamster oocytes by human spermatozoa was markedly raised by successive treatments of the spermatozoa with 5-15 microM ionophore A23187 solutions and a capacitation medium (BWW medium) containing 3.5% HSA. The HSA most effective in inducing capacitation was selected from several kinds of HSA products commercially available. (2) Monospermic fertilization was ensured by inseminating oocytes with highly capacitated spermatozoa at a low concentration for a short time. (3) TC medium 199 was used for postinsemination culture of the eggs. (4) A medium containing podophyllotoxin and vinblastine (0.04 micrograms/ml each) was used to block karyogamy and first-cleavage spindle formation. (5) Chromosome slides were prepared with our gradual fixation-air-dry method instead of Tarkowski's method. Ninety-two to 177 spermatozoa corresponding in number to 43%-79% (mean: 62%) of the inseminated oocytes were successfully karyotyped in each experiment. In spite of above-mentioned quantitative improvements, quality of Q-banding was not necessarily satisfactory in our slides. Improvement of banding technique is an important problem to be solved in our method. Spontaneous incidence of chromosome aberrations was studied in a total of 1,091 spermatozoa obtained from nine semen samples from four donors. Incidences of aneuploidy and structural anomaly were 0.9% (hyperhaploidy, 0.45%; hypohaploidy, 0.45%) and 13.0%, respectively. Structural aberrations included breaks (45.1%), fragments (32.4%), exchanges (21.8%), and deletions (0.7%). Ratio of X-sperm to Y-sperm was 53% to 47%. These results were discussed in comparison with those reported previously.

Maternal ageing and nondisjunction: a comparative study of two chromosomal techniques on the formation of univalents in first meiotic metaphase oocytes of the mouse.

The incidence of univalents was compared between slides prepared according to two clearly different chromosomal methods, i.e. Tarkowski's method and ours, in order to examine whether a univalent pair could be formed artifactually at the first meiotic metaphase (MI). The oocytes used were obtained from young (2-3 months) and old (12-15 months) age groups of both C57BL/6 and dd mice. In Tarkowski's method only a single fixative was used, while in our method three different fixatives were used successively in order to fix oocytes without their being ruptured. Despiralized, fuzzy and loosely associated chromatids were seen frequently in the slides prepared by Tarkowski's method, while such features were seen less frequently in the slides prepared by our method. The incidence of oocytes with univalents in the slides made by Tarkowski's method was much higher than in those made by ours in both age and strain groups (P less than 0.05-0.001). Thus, it was confirmed that the so-called univalents could be produced artifactually. The results did not support the production line hypothesis of Henderson and Edwards (1968) which was based on their observation of an increased incidence of univalents in MI oocytes from aged female mice.