Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes.

BACKGROUND: Although mouse spermatozoa can be freeze-dried without losing their reproductive capacity, the technique needs further improvements to reduce the incidence of chromosomal damage to spermatozoa. Effects of freeze-drying on human spermatozoa are unknown. METHODS: Mouse spermatozoa were suspended in a Tris-buffered EGTA solution briefly (10 min at 37 degrees C) or for 1-7 days at 4 degrees C before freeze-drying. Freeze-dried spermatozoa were maintained for up to 1 year at 4 degrees C before injection. Sperm chromosomes were examined during the first mitosis (cleavage) of zygotes. The ability of sperm to support embryo development was assessed by examining mid-gestation fetuses (Day 14) after transfer of 2-cell embryos to surrogate mothers. Chromosome integrity of freeze-dried human spermatozoa was examined by injecting individual spermatozoa into mouse oocytes which were previously enucleated. RESULTS: When mouse spermatozoa were freeze-dried immediately after suspension in Tris-buffered EGTA solution, only c.40% had normal chromosomes. When the mouse spermatozoa were kept in the same solution for 3-7 days before freeze-drying, 85-95% had normal chromosomes and they were able to support embryo development better than those which were in the solution briefly (P < 0.05). Freeze-dried human spermatozoa well maintained their chromosomes regardless of the duration of pre-freeze-drying incubation of spermatozoa in the Tris-buffered EGTA solution. CONCLUSIONS: Prior incubation of mouse spermatozoa in Tris-buffered EGTA solution for several days makes sperm chromosomes more resistant to freeze-drying. As the consequence, spermatozoa freeze-dried this way support embryo development better than those exposed to Tris-buffered EGTA solution only briefly. Freeze-dried human spermatozoa well maintained their chromosomes without pre-freeze-drying incubation in Tris-buffered EGTA solution.

Fertilizability and chromosomal integrity of frozen-thawed Bryde's whale (Balaenoptera edeni) spermatozoa intracytoplasmically injected into mouse oocytes.

Prior to attempting the in vitro production of embryos in the Bryde's whale (Balaenoputera edeni), we investigated whether spermatozoa can retain the capacity for oocyte activation and pronucleus formation as well as chromosomal integrity under cryopreservation by using intracytoplasmic sperm injection (ICSI) into mouse oocytes. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3-97.4%), and sperm nuclei successfully transformed into male pronucleus within activated ooplasm (87.2-93.6%). Chromosome analysis at the first cleavage metaphase (M) of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, while the percentage of chromosomal normality was significantly reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa due to the increase in structural chromosome aberrations. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development.

A cytogenetic study of in-vitro matured murine oocytes after ICSI by human sperm.

BACKGROUND: The purpose of this study was to investigate the chromosomal complement and developmental potential of in-vitro matured murine oocytes following ICSI by human sperm. METHODS: Heterologous ICSI fertilization between mouse oocytes and human sperm was employed in order to overcome the reduced fertilization rates observed after conventional IVF due to zona hardening during in-vitro maturation, and to assess separately maternal and paternal chromosome complements. Cytogenetic analyses were performed in four types of oocytes: (i) in-vitro matured metaphase II (MII) oocytes; (ii) in-vivo matured MII oocytes; (iii) in-vitro matured oocytes after ICSI; (iv) in-vivo matured oocytes after ICSI. RESULTS: Activation rates after ICSI of in-vitro matured oocytes was lower than that of in-vivo matured oocytes (69.9 versus 97.2%, P < 0.01), and premature chromosomal condensation was only observed in in-vitro matured oocytes. However, there were no significant differences in developmental rates after successful activation between in-vivo and in-vitro matured ICSI oocytes (69.7 versus 76.6%). The incidences of aneuploidy and structural aberrations were similar between the ICSI embryos and non-ICSI (MII) oocytes. Furthermore, the frequency of chromosomal aberrations was not associated with in-vitro or in-vivo maturation. Similar analyses of paternal chromosomes indicated that there were no significant differences in the incidence of chromosomal aberrations between the embryos derived from in-vitro and in-vivo matured oocytes. CONCLUSIONS: These results suggest that in-vitro matured oocytes following ICSI do not lead to an increase in the frequency of aneuploidy and structural aberrations when human sperm are injected into mouse oocytes.

Meiotic stage-dependent induction of chromosome aberrations in Chinese hamster primary oocytes exposed to topoisomerase II inhibitor etoposide.

To investigate the chromosomal effects of topoisomerase II (topo-II)-interactive drugs on mammalian primary oocytes, female Chinese hamsters were treated with etoposide (VP-16) at various intervals pre- and post-human chorionic gonadotropin (hCG) injections. Chromosome analysis of oocytes at metaphase II (M II) showed that treatment with VP-16 at 50h pre-hCG had no effect, but the treatments between 24h pre-hCG and 2h post-hCG often caused structural chromosome aberrations. Although treatment at 4h post-hCG had no effect, subsequent treatments at 6 and 8h post-hCG produced a significant increase in structural chromosome aberrations. No effect was found following treatment at 10h post-hCG. The incidence of aneuploidy following exposure to VP-16 was also dependent on the time of hCG injection. Taking the time course of meiotic progression in primary oocytes following hCG injection and pharmacokinetics of VP-16 into consideration, it is likely that meiotic stages from late dictyate to diakinesis are highly sensitive to VP-16, while stages at dictyate and from metaphase I (M I) to telophase I (telo I) are relatively insensitive to the drug. Moreover, the effect of VP-16 on structural chromosome aberrations and aneuploidy was dose-dependent. Chromosome analysis at M I detected a frequent occurrence of structural chromosome aberrations in treated oocytes. This suggests that structural aberrations may be caused by disruption of cleavable complexes during chromosome condensation. Detection of chromosome bridges during anaphase I/telophase I (ana I/telo I) may support the hypothesis that induction of aneuploidy by VP-16 is due to failure in decatenation of recombinant homologous chromosomes.

Chromosome analysis of human spermatozoa following in vitro exposure to cyclophosphamide, benzo(a)pyrene and N-nitrosodimethylamine in the presence of rat liver S9.

For the purpose of assessing mutagenic effects (clastogenicity) of metabolites derived from chemical mutagens/carcinogens on human sperm chromosomes, spermatozoa were exposed in vitro to cyclophosphamide (CP), benzo(a)pyrene (BP) or N-nitrosodimethylamine (NDMA) for 2h in the presence or absence of rat liver S9, a metabolic activator of these chemicals. After in vitro fertilization between human spermatozoa and zona-free hamster oocytes, chromosome complements of sperm origin were analyzed cytogenetically. In the absence of S9, none of three chemicals (20 microg/ml CP, 200 microg/ml BP and 20mg/ml NDMA) caused a significant increase in spermatozoa with structural chromosome aberrations (8.6, 10.0 and 7.5%), as compared with their matched controls (10.9, 11.0 and 8.5%). In the presence of S9, however, a significant increase in chromosomally abnormal spermatozoa was observed in CP (37.1%, P < 0.001) and BP (31.0%, P < 0.001), indicating that enzymatic activation of CP and BP induced chromosomal abnormalities in human sperm. In contrast, NDMA did not induce chromosome aberrations in human spermatozoa by S9 treatment, although positive results have been observed in somatic cells. The present results on in vitro clastogenicity of CP, BP and NDMA are consistent with the results in previous in vivo studies with murine spermatozoa. Our S9/human sperm chromosome assay seems to be useful for estimation of hereditary risk of chemicals in human. Because most chemicals need metabolic activation to bind to DNA.

Dithiothreitol induces sperm nuclear decondensation and protects against chromosome damage during male pronuclear formation in hybrid zygotes between Chinese hamster spermatozoa and Syrian hamster oocytes.

The present study was undertaken to investigate whether a time lag in sperm nuclear decondensation and male pronuclear formation in the course of development of eggs is associated with any occurrence of structural chromosome aberrations in male genomes of hybrid zygotes between Chinese hamster spermatozoa and zona-free Syrian hamster oocytes. Shortly after insemination, hybrid zygotes were treated with dithiothreitol (DTT) at different concentrations (0.1-10.0 mM) for 30 min to reduce protamine disulphide (S-S) bonds and thereby accelerate sperm nuclear decondensation and male pronuclear formation. The incidence of sperm nuclear decondensation and male pronuclear formation increased with increasing DTT concentrations, indicating that a reduction in S-S bonds effectively induces these cytological events. Chromosomes of male genomes in hybrid zygotes generated by treatment with 1.0 mM, 2.5 mM and 10.0 mM DTT were analysed at the first cleavage metaphase. Incidence of structural chromosome aberrations in each treatment was 34.5%, 27.1% and 24.7%, respectively. There was a significant difference between the incidences with 1.0 mM and 10.0 mM DTT treatment. As the time lag in nuclear decondensation and male pronuclear formation was greatest in the 1.0 mM treatment condition, followed in order by 2.5 mM and 10.0 mM, it is suggested that the lag in sperm nuclear development behind egg development is responsible for structural chromosome aberrations in male genomes of hybrid zygotes.

No induction of chromosome aberrations in human spermatozoa exposed to extremely low frequency electromagnetic fields.

Clastogenic effects of extremely low frequency electromagnetic fields (ELF-EMFs) on human sperm chromosomes were studied using an interspecific in vitro fertilization system with zona-free golden hamster oocytes. Semen samples from healthy men were exposed to ELF-EMFs (50 Hz, 20 mT) for 2 h at 37 degreesC under 5% CO2 in air. The samples were then cryopreserved in liquid nitrogen for shipment to a cytogenetic laboratory. After thawing the samples, motile spermatozoa were collected using a continuous Percoll density gradient centrifugation and then capacitated for in vitro fertilization with hamster oocytes. Sperm-derived chromosomes were analyzed at first cleavage metaphase. The present experiment was performed twice using semen samples from two different donors. In test-1, incidence of spermatozoa that displayed structural chromosome aberrations was 17.0% (35/206) in the exposed group and 20.8% (55/264) in the control group. In test-2, structural chromosome aberrations were observed in 11.1% (13/117) of exposed spermatozoa and 13.8% (13/94) of spermatozoa in the control group. In both tests, there was no significant difference in the incidence of chromosomally abnormal spermatozoa between the exposed group and the control group. Types of aberrations observed and their incidences per spermatozoon in the exposed group were similar to those of the control group. Despite the small sample size, the present results suggest that ELF-EMFs have no clastogenic effect on human sperm chromosomes.

Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation.

This study was undertaken parthenogenetically to activate Chinese hamster oocytes in vitro by chemical stimuli. Oocytes were exposed to five different chemical agents, ethanol (EtOH), strontium chloride (SrCl2), cycloheximide (CHX), phorbol ester (PMA), and ionophore A23187 (IA23). No parthenogenetic activation was observed in the oocytes treated with 8% EtOH for 8-11 min, 1.7 mM and 5.0 mM SrCl2 for 1 hr, 100 microM and 400 microM CHX for 2 hr, and 81 nM and 162 nM PMA for 5 min. In contrast, 89.7% of oocytes parthenogenetically extruded the second polar body in treatment with 3 microM IA23 for 5 min, but only 22.6% of them formed a pronucleus and developed to 2-cell embryos. The remaining ova stopped their cell cycle immediately after completion of the second meiotic division. They had unichromatid chromosomes (monads), which are called MIII chromosomes. Treatment with 5 microM IA23 for 5 min was so deleterious that > 90% of oocytes were degenerated. However, oocyte activation was significantly improved when the treatment with 3 microM IA23 for 5 min was followed by treatment with 8% EtOH for 10 min, 100 microM CHX for 2 hr, 81 nM PMA for 5 min or 3 microM IA23 for 5 min: rates of pronuclear formation were 54.4%, 84.3%, 34.2%, and 54.6%, respectively. More than 80% of pronucleate ova successfully developed into 2-cell stage. Additive treatment with 5 mM SrCl2 for 1 hr had no positive effect on pronuclear formation. Incidences of aneuploidy (4.6%) and structural chromosome aberrations (1.0%) in parthenogenons produced by combined stimuli of IA23 and CHX were not significantly different from those (3.8% and 1.6%, respectively) in female pronuclei of ova fertilized in vitro, showing that combined treatments with IA23 and CHX cause neither nondisjunction at the second meiotic division nor structural aberrations in MII chromosomes. The present technique for parthenogenetic activation of Chinese hamster oocytes may be useful as an assessment system to detect aneugenic and clastogenic effects of mutagens on mammalian oocytes.

Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes.

The chromosome constitution of human spermatozoa was determined after injecting individual spermatozoa into mouse oocytes. Of a total 279 eggs arrested at first cleavage metaphase, 200 (71.7%) were suitable for the analysis of sperm chromosomes. Incidences of spermatozoa with numerical and structural chromosome aberrations were 1.3 and 6.9% respectively in spermatozoa with normal head morphology, showing values comparable with those found in previous studies using the hamster oocyte-human sperm fusion system. The ratio of X- to Y-bearing spermatozoa did not differ significantly from the expected 1:1 ratio. The incidence of structural chromosome aberrations was about four times higher in spermatozoa with amorphous, round and elongated heads (26.1%) than in those with morphologically normal heads, whereas the incidence of aneuploidy was not significantly different between the two groups. No increase in chromosome aberrations was found in spermatozoa with large heads. The same was true for spermatozoa with small heads. Although the sample size used in this study is rather small, the results nevertheless indicate that some morphological abnormalities in the sperm heads are associated with their chromosome defects.

Penetration of concanavalin-A-treated Chinese hamster oocytes by golden hamster spermatozoa in vitro, and chromosome analysis of hybrid 1-cell zygotes.

Pretreatment of zona-free Chinese hamster (CH) oocytes with three kinds of lectin--concanavalin A (Con-A), phytohaemagglutinin-P (PHA) and wheat germ agglutinin (WGA)--was attempted in order to improve penetration by golden hamster (GH) spermatozoa in vitro. Con-A had no significant effect on penetration at 2 micrograms/ml, adequately facilitated oocyte-sperm fusion at 4 micrograms/ml, and caused excessive sperm binding and resultant severe polyspermy at 10 micrograms/ml. Neither PHA nor WGA had positive effects on sperm penetration at any concentrations (2-10 micrograms/ml) examined. Using the Con-A (4 micrograms/ml) pretreatment, high rates of interspecific fertilisation and subsequent chromosome analysis of hybrid 1-cell zygotes were achieved. Among 258 CH oocytes used, 212 (82.2%) were fertilised and 153 (72.2% of fertilised ova) developed to the first cleavage metaphase. Eventually, 132 CH-derived chromosome complements and 153 GH-derived ones were successfully karyoanalysed. Incidences of aneuploidy and structural anomaly were 3.1% and 2.3% in CH complements, and 1.4% and 6.5% in GH complements, respectively. These incidences were not significantly different from those obtained by intraspecific in vivo fertilisation, suggesting that our interspecific in vitro fertilisation system does not cause chromosome aberrations.

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.

In vitro fertilisation of Chinese hamster oocytes by spermatozoa that have undergone ionophore A23187-induced acrosome reaction, and their subsequent development into blastocysts.

To enhance potential use of the Chinese hamster, Cricetulus griseus, in developmental and cytogenetic studies of mammalian gametes and embryos, techniques for in vitro fertilisation and embryo culture were developed in the species. Spermatozoa were recovered from the vasa deferentia of mature males, and incubated in modified TYH medium for 1 h at 37 degrees C under 5% CO2 in air. They were then treated with ionophore A23187 (20 microM) for 10 min to induce the acrosome reaction. Following ionophore treatment, superovulated oocytes were collected from hormonally stimulated females and incubated with the acrosome-reacted spermatozoa for 2 h at 37 degrees C under 5% CO2 in air. In this study, 245 oocytes were used for insemination, and 198 (80.8%) were found to be penetrated by sperm; among them, 194 ova (98.0%) were determined to be monospermic. The monospermic ova were then cultured in TYH supplemented with 1 mM hypotaurine under the same gas phase. Within 30 h of fertilisation, 182 ova (93.8%) cleaved to the 2-cell stage, and subsequently 163 ova (84.0%) developed beyond the 2-cell stage. Thus, obstinate developmental arrest at the 2-cell stage ('2-cell block') was not observed in this species. Ultimately, 65.5% of monospermic ova reached morula to blastocyst stages.

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.

A chromosome painting method for human sperm chromosomes using fluorescent in situ hybridization.

A method of chromosome painting on human sperm chromosomes using fluorescent in situ hybridization (FISH) is introduced. Sperm chromosome slides were prepared after in vitro fertilization of hamster eggs with human spermatozoa. The slides were treated by RNase A before FISH. Chromosome 4 was clearly and specifically painted in a majority of sperm-derived metaphase plates after an application of whole chromosome painting DNA probes of this chromosome. This is the first report of successful painting on human sperm chromosomes.

[A cytogenetic study of human oocytes unfertilized in in-vitro fertilization (IVF)].

A total of 260 unfertilized human oocytes obtained in our IVF program were studied cytogenetically, by a gradual fixation-air drying method proposed by Mikamo and Kamiguchi (1983). The oocytes were obtained from 72 cycles of 52 patients (mean age 32.1). Indications for these patients were 26 cases of tubal factor, 13 of male infertility, 2 of endometriosis and 11 of unexplained infertility. Ovulation induction was performed by hMG-hCG administration with GnRHa pretreatment. Of the 260 unfertilized oocytes, 183 displayed metaphase II and 149 were karyotyped accurately. There were 5 diploids (3.4%), 9 structural anomalies (6.0%) and 15 aneuploids (10.1%) including 8 hyperhaploids (5.4%) and 7 hypohaploids (4.7%). One hundred and twenty-two oocytes out of 149 (81.9%) were found to be normal haploids [23,X]. In 9 aneuploids, 5 showed the loss or gain of monads (so-called predivision), while 4 showed the loss or gain of dyads (so-called nondisjunction). Twenty-three oocytes out of 149 (15.4%) had one or more split dyads. (so-called nondisjunction). Twenty-three oocytes out of 149 (15.4%) had one or more split dyads. Our results were compared with those obtained in previous studies, and the importance of the chromosome preparation method in evaluating aberration frequencies was stressed. Causal mechanisms of aneuploidy, diploidy and structural anomaly were also discussed.

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.