Cleavage Metaphase Research Articles

Toxicity of the Mycotoxin Deoxynivalenol on Early Cleavage of Mouse Embryos by Fluorescence Intensity Analysis.

Deoxynivalenol is a mycotoxin, produced by Fusarium from contaminated corn, wheat, and other grains, that induces multiple effects in humans and animals, including cytotoxic, genotoxic, immunotoxic, and carcinogenic effects. Recent studies show that deoxynivalenol also affects the reproductive system of mammals, including oocyte quality. However, the effects of deoxynivalenol on early embryonic development have not been reported. In this study, fluorescence intensity analysis was used to show that deoxynivalenol disrupted the first cleavage of the zygote. The high deoxynivalenol dose disturbed the movement of the pronucleus after fertilization, while the low deoxynivalenol dose caused aberrant spindle morphology during the metaphase of the first cleavage. Further analysis showed that the reactive oxygen species level increased in the deoxynivalenol-exposed two-cell embryos, indicating oxidative stress. Moreover, deoxynivalenol caused DNA damage in the embryos, as positive γH2A.X signals were detected in the nucleus. These events led to the early apoptosis of mouse embryos, which was confirmed by autophagy. Taken together, our study provides evidence for the toxicity of deoxynivalenol during early embryonic development in the mouse model.

Podophyllotoxin Exposure Causes Spindle Defects and DNA Damage-Induced Apoptosis in Mouse Fertilized Oocytes and Early Embryos.

Podophyllotoxin (PPT) is a kind of lignans extracted from the roots and stems of the genus Podophyllum from the tiller family, and it has been widely used in the treatment of condyloma acuminatum, multiple superficial epithelioma in the clinics. However, PPT has been reported to be toxic and can cause liver defects and other organ poisoning. In addition, emerging evidences also indicate that PPT has reproductive toxicity and causes female reproduction disorders. In this study, we used fertilized oocytes and tried to explore the effects of PPT on the early embryonic development with the mouse model. The results showed that exposure to PPT had negative effects on the cleavage of zygotes. Further analysis indicated that PPT could disrupt the organization of spindle and chromosome arrangement at the metaphase of first cleavage. We also found that PPT exposure to the zygotes induced excessive reactive oxygen species (ROS), suggesting the occurrence of oxidative stress. Moreover, in the PPT-exposed embryos, there was positive γH2A.X and Annexin-V signals, indicating that PPT induced embryonic DNA damage and early apoptosis. In conclusion, our results suggested that PPT could affect spindle formation and chromosome alignment during the first cleavage of mouse embryos, and its exposure induced DNA damage-mediated oxidative stress which eventually led to embryonic apoptosis, indicating the toxic effects of PPT on the early embryo development.

In vivo repair of DNA damage induced by X-rays in the early stages of mouse fertilization, and the influence of maternal PARP1 ablation

The early pronucleus stage of the mouse zygote has been characterised in vitro as radiosensitive, due to a high rate of induction of chromosome-type chromosome abnormalities (CA). We have investigated the repair of irradiation induced double strand DNA breaks in vivo by γH2AX foci and first cleavage metaphase analysis. Breaks were induced in sperm and in the early zygote stages comprising sperm chromatin remodelling and early pronucleus expansion. Moreover, the role of PARP1 in the formation and repair of spontaneous and radiation-induced double strand breaks in the zygote was evaluated by comparing observations in C57BL/6J and PARP1 genetically ablated females. The results confirmed in vivo that the rate of chromosome aberration induction by X-rays was approximately 3-fold higher in the zygote than in mouse lymphocytes. This finding was related to a diminished efficiency of double strand break signalling, as shown by a lower rate of γH2AX radiation-induced foci compared to that measured in most other somatic cell types. The spontaneous frequency of CA in PARP1 depleted zygotes was slightly but significantly higher than in wild type zygotes. Also, these zygotes showed some impairment of the radiation-induced DNA Damage Response when exposed closer to the start of S-phase, revealed by a higher number of γH2AX foci and a longer cell cycle delay. The rate of chromosome aberrations, however, was not elevated over that of wild type zygotes, possibly thanks to backup repair pathways and/or selection mechanisms against damaged cells. When comparing with the literature data on irradiation induced CA in mouse zygotes in vitro, the levels of induction were strikingly similar as was the frequency of misrepair of double strand breaks (γH2AX foci). This result can be reassuring for in vitro human gamete and embryo handling, because it shows that culture conditions do not significantly affect double strand DNA break repair.

Characterization of chromosomal damage accumulated in freeze-dried mouse spermatozoa preserved under ambient and heat stress conditions

Structural chromosome aberrations and DNA damage generated in freeze-dried mouse spermatozoa were investigated. Freeze-dried sperm samples were preserved at 4, 25 and 50°C for short duration (1 day to 2 months) and at 25°C for long duration (2 years). The spermatozoa were injected into mouse oocytes to analyse the chromosomes of the zygotes at the first cleavage metaphase. Chromosome break of the chromosome-type aberrations was the most common type of structural chromosome aberrations observed in all freeze-dried samples. The frequency of chromatid exchanges rapidly increased in freeze-dried spermatozoa preserved at 50°C for 1-5 days. The frequency of chromatid-type aberrations (break and exchange) gradually increased in freeze-dried spermatozoa preserved at 25°C for up to 2 months. Alkaline comet assay revealed significant migration of damaged DNA accumulated in freeze-dried spermatozoa preserved at 50°C for 3 days and 25°C for 2 years. However, no DNA damage was detected using the same sperm samples by neutral comet assay, which can detect mostly DNA double-strand breaks in cellular DNA. These results suggest that DNA single-strand breaks were accumulated in freeze-dried spermatozoa preserved under ambient or heat conditions, and then chromatid-type aberrations, especially the chromatid exchanges, were formed via post-replication repair system in zygotes.

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.

381 FERTILIZABILITY AND CHROMOSOMAL INTEGRITY OF FROZEN - THAWED BRYDE's WHALE (BALAENOPTERA EDENI) SPERMATOZOA INTRACYTOPLASMICALLY INJECTED INTO MOUSE OOCYTES

In this study, we applied intracytoplasmic sperm injection (ICSI) to mouse oocytes to evaluate the fertilizability and chromosomal integrity of the three types of frozen–thawed Bryde's whale spermatozoa. B6D2F1 female mice (7–11 weeks of age) were superovulated by injections of PMSG followed by hCG 48 h later. The oocytes recovered from oviducts between 14 and 16 h after hCG injection were denuded of their cumulus cells. Sperm samples were obtained from a Bryde's whale (Balaenoptera edeni) captured under the Japanese Whale Research Program with Special Permit in the Western North Pacific between May and August 2003 (presumptive feeding season). The whale was killed by an explosive harpoon which has been recognized as the best humane method for whales by the International Whaling Commission (IWC) and stipulated by Schedule III (Capture) of the International Convention for the Regulation of Whaling. Spermatozoa collected from vasa deferentia were cryopreserved. Frozen Bryde's whale spermatozoa were thawed at 37�C and washed with HEPES-TYH by centrifugation at 500g for 5 min. Motile and immotile spermatozoa were obtained, and some spermatozoa in HEPES-TYH were refrozen without cryoprotectant at -20�C to be completely killed. Within 24 h, they were thawed at 37�C and prepared for ICSI. Comparison of group values was performed by either Fisher's exact probability test or chi-square test where necessary. Differences at P d 0.05 were considered significant. Chromosomal normality was determined by analyses of karyotyped haploid chromosomes (n = 22) of the whale sperm. 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 pronuclei within activated ooplasm (87.2–93.6%). Chromosome analysis at the first cleavage metaphase of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, whereas the percentage of chromosomal normality was significantly (P ≤ 0.001) reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa, due to the increase in structural chromosome aberrations such as chromosome fragments. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development. Furthermore, we have shown that chromosomal analysis of whale spermatozoa is a useful technique for measuring the influences of marine pollution on reproduction in cetacean species that occupy the top niche in the marine ecosystem.

生殖細胞および培養細胞を用いた遺伝毒性試験法の開発と国際標準化への貢献

Biological effects of genotoxic chemicals are classified into two major categories depending on whether the target organs contain germ cells or just somatic cells. Genotoxicity in germ cells may cause heritable damage in offspring. In the case of somatic cells, genetic damage in a single targeted cell may result in cancer through a multistep process. In this review, I present our data obtained from both areas of germ cell and somatic cell genotoxicity. For studies in male mice, alkylating agents are known to induce dominant lethal mutations in the late spermatids and mature sperm. In studying spermatogenesis and oogenesis in mice, we found that there were stage-specific responses to chemicals. Also, we clarified that such responses to induce heritable translocations closely relate to the types of chromosome aberrations induced in the repair-deficient late spermatid and mature sperm stages. Finally the high incidence of heritable translocations in F1 mice originated from the chromosome type aberrations induced in the first cleavage metaphases of fertilized eggs. Thus, germ cell stages in the mature sperm and oocytes just after copulation were sensitive to mutagens according to the results of chromosome analysis at the first cleavage.Transformation assays have been used for detection of possible carcinogens, not only initiators but also promoters. In our previous studies, we have tried to use transformation assays such as Syrian hamster embryo (SHE) cells and rat tracheal epithelial (RTE) cells as primary culture cells and also Balb/c 3T3 cells and Bhas42 cells as immortalized cell lines. As an example of transformation assays using primary cells, RTE cell transformation assay system is described. In this study, we could detect a promotion activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by the enhancement of MNNG-induced transformation. This promotional activity may be attributed to a promotional effect, a comutagenic action, or a modulation of cell proliferation and/or differentiation mediated through the TCDD receptor. On the other hand, Balb/c 3T3 assay involves immortalized mouse fibroblast cell lines, in which the number of foci in a monolayer are scored. We have performed an inter-laboratory validation study of the improved transformation assay reported by Drs. Tsuchiya and Umeda with members of NGCS (Non-genotoxic Carcinogen Study Group) under JEMS (Japanese Environmental Mutagen Society). In this study, we gained confidence in the usefulness of the modified two-stage transformation assay with Balb/c 3T3 cells. Bhas42 cells have been established by transfection of v-Ha-ras to Balb/c 3T3 cells by Sasaki et al., and we found that Bhas42 cells were extremely susceptible to TPA-type promoters. Recently, NGCS have started a validation study to evaluate the Bhas42 cell assay system to detect promoters using the modified method reported by Omori et al. This cell transformation assay has advantages over the original Balb/c 3T3 cell assay, particularly regarding sensitivity, test period and simplicity.

Chromosome analysis of mouse zygotes after injecting oocytes with spermatozoa treated in vitro with green tea catechin, (−)-epigallocatechin gallate (EGCG)

The cytogenetic effects of (−)-epigallocatechin gallate (EGCG) on mouse spermatozoa were studied in vitro using an intracytoplasmic sperm injection (ICSI) technique. Spermatozoa were collected by the swim-up method and treated with EGCG at 1 μM and 10 μM. When motile, EGCG-treated spermatozoa were injected into oocytes, structural chromosome aberrations (SCAs) at the first cleavage metaphase did not increase significantly. However, a majority of immotile spermatozoa treated with 10 μM EGCG had the following abnormalities: pronuclear arrest (11% of activated oocytes), degenerated sperm chromatin (chromosome) mass (30% of activated oocytes) and occurrence of structural chromosome aberrations (57% of analyzed metaphases). The incidence of these abnormalities suggests that immotile spermatozoa were susceptible to EGCG, and that the damage of sperm chromatin was accelerated in immotile spermatozoa by 10 μM EGCG treatment.

Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs γ-ray irradiation

This study demonstrated that freeze-dried mouse spermatozoa possess strong resistance to 137Cs γ-ray irradiation at doses of up to 8 Gy. Freeze-dried mouse spermatozoa were rehydrated and injected into mouse oocytes with an intracytoplasmic sperm injection (ICSI) technique. Most oocytes can be activated after ICSI by using spermatozoa irradiated with γ-rays before and after freeze-drying. Sperm chromosome complements were analyzed at the first cleavage metaphase. Chromosome aberrations increased in a dose-dependent manner in the spermatozoa irradiated before freeze-drying. However, no increase in oocytes with chromosome aberrations was observed when fertilized by spermatozoa that had been irradiated after freeze-drying, as compared with freeze-dried spermatozoa that had not been irradiated. These results suggest that both the chromosomal integrity of freeze-dried spermatozoa, as well as their ability to activate oocytes, were protected from γ-ray irradiation at doses at which chromosomal damage is found to be strongly induced in spermatozoa suspended in solution.

Chromosome analysis of human spermatozoa with morphologically abnormal heads by injection into mouse oocytes.

Aim: The risk of chromosome aberrations in human spermatozoa with an abnormal head shape was examined using intracytoplasmic sperm injection into mouse oocytes. Methods: Human spermatozoa with small, large, pointed or elongated head shape from a fertile donor were injected into mouse oocytes, and the hybrid oocytes were cytogenetically analyzed at the first cleavage metaphase. Results: The oocyte activation rate was significantly lower in hybrid oocytes injected with pointed (90.2%) or elongated spermatozoa (94.1%) than with normal spermatozoa (99.0%). However, the frequency of intracytoplasmic sperm injection oocytes at the first cleavage metaphase did not differ among the sperm groups (71.8-77.2%). No difference in the incidences of aneuploidy (1.8, 2.6 and 1.4%), diploidy (0, 0 and 0%) and structural chromosome aberrations (6.3, 10.4 and 8.6%) was observed between small or pointed spermatozoa and normal spermatozoa. Only a small population of large spermatozoa (3.7%) was diploidy. Elongated spermatozoa showed significantly frequent structural chromosome aberrations (33.3%) as compared with normal spermatozoa. Conclusion: The results reveal some interesting details of the mechanism of sperm nuclear condensation: small sperm size is attributed to the problem of nuclear condensation, not a decrease in chromosomes. Diploidy prevents sperm nucleus from condensing, resulting in a large sperm head. Elongation of sperm nucleus causes DNA lesions leading to structural chromosome aberrations. (Reprod Med Biol 2004; 3: 147-152).

Frequent structural chromosome aberrations in immotile human sperm exposed to culture media.

The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.

Ability to activate oocytes and chromosome integrity of mouse spermatozoa preserved in EGTA Tris–HCl buffered solution supplemented with antioxidants

Potential methods for cryopreservation of mouse spermatozoa are freeze-drying, desiccation, and suspension in EGTA Tris–HCl buffered solution (ETBS: 50 mM NaCl, 50 mM EGTA, and 10 mM Tris–HCl). To determine the duration that mouse spermatozoa suspended in ETBS-based solutions could retain their normal characteristics without freezing, spermatozoa collected from the cauda epididymis were suspended in ETBS or in ETBS supplemented with the antioxidants, dimethyl sulfoxide (DMSO), or dl-α-tocopherol acetate (Vitamin E acetate; VEA) diluted in DMSO, then held at ambient temperature (22–24 °C) for up to 9 days. When oocytes were injected with spermatozoa preserved in ETBS alone, activation rates of oocytes and chromosome integrity at the first cleavage metaphase decreased at 1 day ( P<0.001) and 2–4 days ( P<0.01) following treatment. When oocytes were injected with spermatozoa preserved in ETBS supplemented with DMSO or VEA/DMSO, chromosome integrity did not decrease significantly (through 9 days of preservation). Although DMSO maintained sperm chromosome integrity more effectively than VEA/DMSO up to 2–4 days (91 and 67%, normal karyotypes in DMSO and VEA/DMSO, respectively), VEA/DMSO helped to maintain the ability of spermatozoa to activate oocytes, but did not enhance the maintenance of sperm chromosome integrity. These results suggested that deterioration of spermatozoa preserved in ETBS alone was delayed by supplementation with antioxidants.

An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach ( Misgurnus mizolepis)

Blocking the first mitotic cleavage of the zygote is a key tool for chromosome-set manipulations in fish. We developed an improved method for inducing tetraploidy by blocking the mitosis with a combination of heat shock at 40.5 °C for 1, 2, or 3 min followed by cold shock at 1.5 °C for 30, 45, or 60 min. When applied during the first cleavage metaphase of mud loach ( Misgurnus mizolepis) zygotes, the optimal combination was heat for 2 min followed by cold for 45 min. At 1 month, the frequency of 4N survivors and the yield from total eggs fertilized was 55.7 and 14.4%, respectively, compared to heat shock alone with 20.0% efficiency and 3.6% yield. The effectiveness of the procedure was confirmed by diploid mitotic gynogenesis using transgenic markers. The overall yield of homozygous diploids, 34.0%, was better than that for single heat shock, 17.3%. The tetraploids and homozygous diploids had higher early mortality than normal diploid controls. However, at 1 month, the viability of the tetraploids was the same as normal diploids. For gynogenetic diploids, the survival was similar to normal diploids after 3 months. The high efficiency of this new protocol extends the opportunity to study polyploidy in basic and applied research.

Avian chromosomes can be examined by injection of erythrocyte nuclei into mouse oocytes.

Avian chromosomes can be examined by injection of erythrocyte nuclei into mouse oocytes.

A detailed cytogenetic analysis of large numbers of fresh and frozen-thawed human sperm after ICSI into mouse oocytes.

Since information about chromosome aberrations in micro-manipulated sperm is still inadequate, cytogenetic analysis was performed on large numbers of fresh and frozen-thawed (FT) human sperm after injection into mouse oocytes. The effects of the ICSI procedure on oocytes are also discussed based on analysis of the mouse chromosome complements. After the injection of fresh and FT human sperm into mouse oocytes, chromosomes of the hybrid oocytes were analysed at first cleavage metaphase. Incidences of the hybrid oocytes at the first cleavage metaphase were significantly different between fresh (71.5%) and FT sperm groups (80.1%) (P < 0.05). The chromosome analysis of 477 fresh and 141 FT sperm showed no difference in the incidences of aneuploidy (1.6/0.7%), structural aberrations (8.8/7.8%) or diploidy (0.0/0.0%) between these categories. The cytogenetic result did not differ from our previous result using IVF between human sperm and hamster oocytes. In an additional cytogenetic study on 615 mouse chromosome complements, the incidence of diploidy (5.4%) was significantly higher than those (0.3-2.8%) in the previous mouse cytogenetic studies, and the hybrid oocytes with no mouse chromosomes (2.0%) existed. This result suggests that the ICSI procedure induces no sperm chromosome aberrations but increases numerical aberrations in oocyte chromosome complements.

Abnormal chromosome migration and chromosome aberrations in mouse oocytes during meiosis II in the presence of topoisomerase II inhibitor ICRF-193

Using a mouse parthenogenetic system, effects of ICRF-193, a noncleavable complex-forming topoisomerase II inhibitor, on female meiosis II chromosomes and pronuclear chromosomes were studied. Eggs were exposed to the inhibitor (10 μM) at various times after parthenogenetic stimulation, and chromosomes of them were analyzed at the first cleavage metaphase. When eggs were exposed to the inhibitor during the period from metaphase II to anaphase II, a significant increase in incidences of structural chromosome aberrations (51.1% versus 1.3% in the control) and aneuploidy (30.3% versus 0.7% in the control) was found. Structural chromosome aberrations were observed in 10–20% of eggs following treatments during telophase II, but there was no increased incidence of aneuploidy in treatments during this meiotic stage. When pronuclear eggs at S phase were targeted by the inhibitor, no significant increase in chromosome aberrations was found. Interestingly, when chromatids moved to each pole during anaphase II in the presence of ICRF-193, most of them oriented their centromeres toward the spindle equator as if moving backwards. Moreover, lagging chromatids with the centromeres present were observed in more than 50% of treated eggs. However, chromosomal bridges that resulted from chromosome stickiness did not appear in any egg. These findings indicate that ICRF-193 can induce structural chromosome aberrations and aneuploidy in mouse secondary oocytes in meiotic stage-dependent manner. The induction of aneuploidy is due to disruption of the separation of sister centromeres at anaphase II. There appears to be mechanism(s) other than cleavable complex formation or chromosome stickiness behind the induction of structural chromosome aberrations by ICRF-193.

Sonication per se is not as deleterious to sperm chromosomes as previously inferred.

Although sonication is a simple way to immobilize ("kill") spermatozoa prior to injection into oocytes, this has been thought to be destructive to sperm chromosomes. Mouse and human spermatozoa were immobilized by sonication and kept in various media for up to 2 h, then their nuclei were individually injected into mouse oocytes for the analysis of chromosomes at the first cleavage metaphase. In both the mouse and human, incidence of structural chromosome aberrations was much higher in the spermatozoa sonicated and stored in Biggers-Whitten-Whittingham medium for 2 h at 37.5 degrees C than in those stored for 5 min in the same medium. We concluded, therefore, that it is not sonication per se but a prolonged exposure of sperm nuclei to extracellular milieu that is detrimental to sperm chromosomes. The incidence of structural chromosome aberrations of mouse and human spermatozoa was significantly reduced when the spermatozoa were sonicated and stored in K(+)-rich nucleus isolation medium containing EDTA. This suggests that sperm chromosome degradation following sperm immobilization by sonication is partly due to detrimental effects of a Na(+)-rich medium and of DNase on sperm chromatin. Ideally, it should be possible to prepare artificial media that maintain the integrity of sperm chromosomes for many hours after immobilization.

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.

Chromosome analysis of BALB/c mouse spermatozoa with normal and abnormal head morphology.

To assess the relationship between mouse sperm head morphology and karyotype, sperm heads with either a normal or an abnormal morphology were injected individually into enucleated mouse oocytes that were karyotyped at the metaphase of the first cleavage. BALB/c male mice that produce an unusually high proportion of morphologically abnormal spermatozoa were used as sperm donors. Abnormal karyotypes were found in a significantly higher proportion of eggs injected with severely misshapen sperm heads (36-38%) as compared to those injected with normal and quasi-normal heads (15-21%) (p < 0.01). Most karyotype abnormalities were structural rather than numerical, the most common being breaks and exchanges of chromosome type in both normal and abnormal spermatozoa.

Improvement of male pronuclear formation during cross-fertilization between Chinese hamster spermatozoa and Syrian hamster oocytes by nocodazole, and chromosome analysis of hybrid zygotes

During cross-fertilization between Chinese hamster spermatozoa and Syrian hamster oocytes, incorporated sperm heads frequently fail to develop into male pronuclei, whereas the group of oocyte chromosomes develop into female pronuclei. The present study applies this cross-fertilization system to the cytogenetic investigation of mammalian hybrid embryos. Immediately after insemination, oocytes were exposed to 0.1 μg/ml nocodazole for 1 hr (1 hr group) or 2 hr (2 hr group), then further cultured. Although the rates of sperm penetration in the 1 hr (48.0%) and 2 hr (75.8%) groups were significantly lower than that in the control group (89.8%), the ratios of male pronuclear formation were higher in both exposed groups (79.4% and 74.2%, respectively) than in the control group (10.6%). These results were apparently due to sperm head decondensation induced during the meiotic arrest of oocytes at metaphase II by nocodazole. Chromosomes of hybrid zygotes obtained after nocodazole exposure were analyzed at the first cleavage metaphase. The incidence of structural chromosome aberrations in the Chinese hamster genome of hybrid zygotes was high in the control (42.1%) and 1 hr (48.8%) groups. This incidence was reduced to 14.4% in the 2 hr group. Because the lag of sperm head decondensation behind the second meiotic division of oocytes was greater in the control and 1 hr groups than in the 2 hr group, untimely sperm head decondensation may be implicated in occurrence of structural chromosome aberrations in the male genomes of hybrid zygotes.