Additional evidence of the formation of unbalanced embryos in cattle with the 7/21 Robertsonian translocation

To confirm the effect of the 7 21 Robertsonian translocation on fertility in Japanese Black Cattle, cytogenetic studies were performed on embryos collected from the following 3 mating groups: normal bull cross normal cow, translocation carrier bull cross normal cow, and normal bull cross translocation carrier cow. All the analyzable embryos showed normal chromosome complements when the parents had a normal karyotype. In the group sired by the 7 21 translocation heterozygous bulls, a total of 56 embryos had metaphases suitable for chromosome analyses. Out of these embryos, 28 had normal chromosome complements and 25 were embryos with a balanced karyotype. However, 3 (5.4%) were monosomic and trisomic embryos, presumably resulting from the fertilization of normal ova by aneuploid spermatozoa. Unbalanced embryos were also observed in the chromosome analyses of embryos derived from the 7 21 translocation heterozygous cows. These results suggest that the 7 21 translocation in the heterozygous state may be associated with a slight reduction in reproductive efficiency.     

Increase of ICSI efficiency with hyaluronic acid binding sperm for low aneuploidy frequency in pig

The present study was designed to evaluate the ability of hyaluronic acid binding sperm (HABS) in increasing the efficiency of intracytoplasmic sperm injection (ICSI) in terms of the production of chromosomally normal porcine embryos. Porcine embryos were produced by in vitro fertilization (IVF), ICSI and ICSI using hyaluronic acid binding sperm (ICSI-HABS). Chromosome aneuploidy in sperm and embryos was evaluated using chromosome 1 submetacentric probe for fluorescence in situ hybridization (FISH) analysis. No significant differences were observed in the blastocysts rates (18.6, 23.6 and 23.8%) and cell numbers (61.8+/-12.5, 55.5+/-7.3 and 59.3+/-9.6) among embryos derived from IVF, ICSI, and ICSI-HABS. However, the frequency of normal diploidy in ICSI-HABS (75.5%) was significantly higher (P<0.05) than that in IVF (57.0%) and ICSI (68.2%). Embryos from ICSI-HABS showed significantly lower chromosome abnormality rate (P<0.05). Both ICSI and IVF embryos showed higher rates of polyploidy, and hence chromosomally abnormal embryos, in comparison to ICSI-HABS embryos. In addition, we investigated the chromosomal complement of porcine spermatozoa by FISH. The rate of chromosome number abnormality in porcine sperm was found to be 6.25% (70/1120). Thus, we conclude that the use of hyaluronic acid binding sperm is superior to morphological sperm selection for ICSI in producing chromosomally normal embryos and increasing the ICSI efficiency by lowering the aneuploidy frequency. Our results indicate that the selection of normal sperm with hyaluronic acid binding assay might help to reduce the early embryonic mortality due to chromosomal aneuploidy thereby increasing the success rate of embryo transfer technology in pigs.     

Contribution of chromosomal imbalance to sperm selection and pre-implantation loss in translocation-heterozygous Chinese hamsters

Chinese hamster stocks with various structurally abnormal chromosomes have been produced by X irradiation. Among these stocks, 18 with various reciprocal translocations were used to investigate the participation of unbalanced gametes in fertilization and the development of unbalanced embryos. Among males as well as females heterozygous for the same translocation, there is no difference in the frequency of each disjunctional class. The participation of chromosomally unbalanced gametes in fertilization was investigated by chromosomal analysis of meiotic cells in heterozygotes for the 18 reciprocal translocations and pronuclei of fertilized ova obtained from crossing these heterozygotes. Compared with the expected frequencies from MII scoring, the frequencies of male pronuclei having a common deficiency of chromosome 1 (1q17-->1q42) or chromosome 3 (3p23-->3q31) decreased significantly in one-cell embryos. However, the frequencies of male pronuclei with other abnormalities were all consistent with those expected from MII scoring. In contrast, the frequencies of female pronuclei with any karyotype including the same abnormalities as those decreased in male pronuclei from the translocation heterozygotes were all consistent with those estimated from MII scoring. These results revealed clearly that most gametes with nullisomies as well as disomies for any chromosomal segments may participate in fertilization, whereas only male gametes nullisomic for certain segments of chromosomes 1 and 3 failed to participate in fertilization. The zygotic selection of chromosomal imbalance was also investigated by direct chromosomal and morphological analyses of preimplantation embryos from crosses between karyotypically normal females and male heterozygotes from the 18 stocks with various reciprocal translocations. These analyses revealed that some embryos were arrested in development at the two-cell stage. The karyotype of these two-cell embryos had a common deficiency in a segment of chromosome 1 or chromosome 2. Embryos with partial monosomy including chromosomes 1, 3, 4 and 5 showed arrested development at four- to eight-cell stages. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of segments of chromosomes 1p, 1q, 2q, 5q, 7q and 8, had fewer blastomeres than karyotypically normal and balanced embryos. The homology between Chinese hamster and mouse chromosomes relating to abnormal embryogenesis at early stages has been partially confirmed from reported maps of chromosomes. The Chinese hamster is useful for further cytogenetic studies during the stages of meiosis and early embryogenesis.     

Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization

Karyotypic studies of aborted fetuses have been used to draw the inference that the proportion of conceptuses with chromosome abnormalities is very high. Fluorescent in situ hybridization (FISH) studies of blastomeres from early cleavage embryos have provided some support for this inference but they are limited to the study of a few chromosomes. We describe the novel application of comparative genomic hybridization (CGH) to the study of numerical and structural abnormalities of single blastomeres from disaggregated 3-day-old human embryos. CGH results were obtained for 63 blastomeres from 12 embryos. Identification of all chromosomes with the exception of chromosomes 17, 19, 20 and 22 was possible. The embryos divided into four groups: (1) embryos with a normal CGH karyotype seen in all blastomeres; (2) embryos with consistent aneuploidy suggesting meiotic non-disjunction had occurred; (3) embryos that were mosaic generally with one or more cells showing aneuploidy for one or two chromosomes but some with cells showing extensive aneuploidy; and (4) one embryo with extensive aneuploidy in all blastomeres. The extensive aneuploidy in group 4 is interpreted as corresponding to the random aneuploidy seen in "chaotic" embryos reported by using interphase FISH. Partial chromosome loss and gain following chromosome breakage was observed in one embryo. Our analysis provides basic biological information on the occurrence of constitutional and post-zygotic chromosome abnormalities in early human embryos. Used in conjunction with embryo biopsy, diagnostic CGH should allow the exclusion of a proportion of embryos that appear normal but that have a poor probability of survival and, therefore, may improve the implantation rate after in vitro fertilization.     

Chromosomal abnormalities in Day-6, in vitro-produced pig embryos

A cytogenetic study was undertaken to quantify, by chromosomal karyotyping, the incidence and type of chromosomal abnormalities present in Day-6 in vitro-produced (IVP) porcine embryos. Morphologically normal Day-6 blastocysts (n=318) were fixed and grouped into six classes according to the number of total cells (from < or =20 to 61-70). Of 248 embryos suitable for analysis, 97 (39.1%) displayed chromosomal abnormalities. The abnormalities included haploidy (9.3%), polyploidy (71.1%) and mixoploidy (19.6%). Within polyploid embryos, triploidy and tetraploidy showed the highest incidence (56.5 and 27.5%, respectively); among mixoploid embryos, diploid-triploid embryos (2n/3n) were prevalent (36.8%). Overall, the mean cell number was 34.3 +/- 12.1 and the mitotic index was 8.6 +/- 6.1. Chromosomally abnormal embryos had fewer (P<0.01) total cells compared to normal (2n) embryos (31.8 +/- 1.3 versus 35.9 +/- 1.0). In addition, the incidence of polyploidy decreased as the number of cells increased, while that of mixoploidy did not differ. These data indicate that polyploidy affects a large percentage of IVP porcine embryos capable of developing to blastocysts and the incidence of chromosomal abnormalities is much higher than that reported previously in in vivo embryos in this species. Given the ability of morphologically normal embryos with an abnormal chromosome complement to undergo preimplantation development in vitro, and the inability to identify blastocysts with abnormal karyotype without cytogenetic analysis, careful consideration should be given to factors affecting ploidy of IVP embryos, especially the incidence of polyspermic fertilization, when evaluating criteria of a porcine in vitro embryo production scheme.     

Aneuploidy in the human blastocyst

Studies of human cleavage stage embryos, 3 days after fertilization of the oocyte, have revealed remarkably high levels of chromosome abnormality. In addition to meiotic errors derived from the gametes, principally the oocyte, mitotic errors occurring after fertilization are also common, leading to widespread chromosomal mosaicism. The prevalence of chromosome anomalies in embryos may explain the relatively poor fertility and fecundity in humans and the low success rates of assisted reproductive treatments (e.g., IVF). While much is known concerning the incidence of aneuploidy during the first 3 days following fertilization, it is only in the last couple of years that large numbers of embryos at the final stage of preimplantation development, the blastocyst stage, 5 days after fertilization, have been subjected to detailed analysis. Here we discuss the latest data from the comprehensive cytogenetic analysis of blastocysts. These findings indicate that the majority of selection against chromosome abnormalities does not occur until the time of implantation or shortly after, with aneuploidy typically affecting more than 50% of blastocysts. Additionally, clinical results presented suggest that screening of blastocyst stage embryos for chromosome abnormality, with preferential transfer to the uterus of those found to be euploid, may help to improve the success rates of assisted reproductive treatments.     

Transmission of chromosomal abnormalities: participation of chromosomally unbalanced gametes in fertilization and early development of unbalanced embryos in the Chinese hamster

Using 14 Chinese hamster stocks with various reciprocal translocations, chromosomally unbalanced gametes were produced and used to investigate the participation of the unbalanced gametes in fertilization and the development of unbalanced embryos. The selection of chromosomally abnormal gametes during fertilization was investigated by the chromosomal analysis of meiotic cells in heterozygotes for the 14 reciprocal translocations and pronuclei of fertilized ova obtained from crossing these heterozygotes. Compared with the expected frequencies from meiotic metaphase II (MII) scoring, the frequencies of male pronuclei having commonly a deficiency of chromosome 1 (q14-->q42) or chromosome 3 (p23-->q31) in one-cell embryos decreased significantly. However, the frequencies of male pronuclei with other abnormalities were all consistent with those expected from MII scoring. In contrast, the frequencies of female pronuclei with any karyotype including the same ones, as those decreased in male pronuclei from the translocation heterozygotes were all consistent with those estimated from MII scoring. These results suggest that gametes with nullisomies as well as disomies for any chromosomal segments may mostly participate in fertilization, whereas some sperm nullisomic for the specific segments of chromosomes 1 and 3 may fail to fertilize. On the other hand, the zygotic selection of chromosomal imbalance was investigated by direct analyses of pre-implantation embryos from crosses between chromosomally normal females and male heterozygotes from the 14 stocks with various reciprocal translocations. The chromosomal and morphological analysis revealed that some embryos were arrested in development at the two-cell stage and their common abnormality was partial monosomy for chromosome 1 or 2. Embryos with partial monosomy including chromosomes 1, 3 and 4 showed arrested development at four-eight-cell stages. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of other segments, such as chromosomes 1p, 2q, 5q and 8, had fewer blastomeres than karyotypically normal and balanced embryos. The homology between the mouse and the Chinese hamster chromosomes relating to the developmental abnormalities at early stages was partially confirmed.     

Failure of chromosomally abnormal sperm to participate in fertilization in the Chinese hamster.

The selection of chromosomally abnormal gametes was investigated in the Chinese hamster by direct chromosome analysis of meiotic cells and one-cell embryos obtained from crossing heterozygotes for two reciprocal translocations, T(1;3)7Idr and T(1;3)8Idr. Expected frequencies of male and female gametes with different chromosome constitutions were estimated by scoring of secondary meiotic metaphase (MII) cells in the translocation heterozygotes. The frequency of gametes with each karyotype that participated in fertilization was investigated in pronuclei from translocation heterozygotes in one-cell embryos obtained from crossing the heterozygotes with karyo-typically normal animals. Compared with the expected frequencies from MII scoring, the frequencies of male pronuclei having some karyotypes in one-cell embryos decreased significantly. The karyotypes of male pronuclei showing a decreased frequency were commonly characterized by a deficiency of the long-arm segment of chromosome 1 (q13----qter) or by a deficiency of almost the whole arms of chromosome 3. On the other hand, the frequencies of female pronuclei with the same karyotypes were all consistent with those estimated from MII scoring. These results suggest that sperm nullisomic for certain segments of some chromosomes may fail to participate in fertilization.     

Status of oocytes and embryos of X-autosome translocation carrier sows

The impact of an X-autosome translocation t(Xp+; 14q-), on ovulation, fertilization and embryo survival in carrier sows, was examined and compared with these parameters of normal sows. Corpora lutea counts during week-2 and week-4 of gestation were similar in normal and carrier sows (14.4 +/- 1.36 and 15.5 +/- 2.18) although embryo recovery (11.0 +/- 1.87 and 6.0 +/- 1.47) was lower than that from normal sows (12.8 +/- 1.46 and 11.5 +/- 0.87), at these stages. Among the embryos karyotyped from the week-2 embryos of carrier sows, 42% were normal, 26.4% were carriers and 31.6% were of unbalanced chromosome make-up, and of the week-4 embryos of carriers, 33.3% were normal, 57.1% were carriers and 9.1% were chromosomally unbalanced females. The preponderance of females among the unbalanced embryos recovered at week-2 of gestation (11_ and 1_) and the total absence of males among those recovered at week-4, suggest that oocytes with unbalanced chromosome constitution are eliminated before week-2 of gestation if they are fertilized by Y bearing sperm, and that the unbalanced oocytes fertilized by X bearing sperm survive up to the peri-attachment stage even though all chromosomally unbalanced embryos are eliminated before term regardless of their sex.     

Cytogenetics of human oocytes,zygotes, and embryos after in vitro fertilization

Summary Chromosome errors, inherited or arising de novo during gametogenesis and transmitted at fertilization to the conceptus, may be a major cause of embryonic mortality. The in vitro fertilization and embryo transfer (IVF/ET) procedure provides extra material — oo-cytes, zygotes, and embryos — to investigate the contribution of chromosomal abnormality to implantation failure. This paper reviews the results of cytogenetic studies on such material. Estimates from a total of 1120 oocytes from 11 studies give an overall proportion of chromosomal abnormality of 35%. Single and multiple nullisomies and disomies are found, involving nonrandom chromosome gain or loss. Hypohaploid complements are more frequent than hyperhaploid complements. The higher rate of chromosome loss of hypohaploid karyotypes was found to be largely artifactual. The estimated overall frequency of aneuploidy is 13%. In embryos the level of chromosomal abnormality is 23%–40%. Errors of fertilization are responsible for a substantial number of triploid embryos, many of which develop into mosaics. Factors extrinsic to the conceptus, such as infertility, advanced maternal age, and ovarian hyperstimulation, may increase the level of chromosomal abnormality. More refined methods for accurately recognizing and selecting chromosomally normal embryos for transfer are needed to improve the success rate of this reproductive technology.     

Embryonic loss in superovulated cattle caused by the 1;29 Robertsonian translocation

The effect of the 1;29 Robertsonian translocation on fertility was studied using embryos resulting from matings of nine carrier cows and two carrier bulls. Embryos were collected from the following three mating groups utilizing superovulation: normal bull cross normal cow, normal bull cross translocation carrier cow, and translocation carrier bull cross normal cow. The proportion of ova which were fertilized did not vary among the groups, indicating that fertilization rates were not affected by the translocation. The translocation cows did yield fewer embryos on average than did cows with normal karyotypes, which may suggest ovulation rates are reduced (at least after superovulation attempts) in cattle carrying the 1;29 translocation. Twenty of 39 embryos successfully karyotyped had abnormal chromosome complements. All four of the theoretically predicted karyotypes and two additional abnormal combinations were found. Eight of 39 (20.5%) embryos karyotyped had unbalanced karyotypes which would have resulted in embryonic loss. The proportion of embryos with unbalanced karyotypes, was slightly higher when the cow (36%) carried the translocation than when the bull (19%) did. Results of this study indicate that fertility is impaired due to the presence of this translocation. The major loss in reproductive potential appears to be due to embryonic loss rather than fertilization failure.     

银鲫精子在两性融合生殖鱼类卵质中的受精特征

红鲤(♀)×银鲫(♂)的杂种胚胎均不能成活。受精细胞学研究结果表明,雌核发育银鲫的精核在两性融合生殖的鲤鱼卵质中能转化为雄性原核,并和雌性原核融合成合子核。但在卵裂开始后,可观察到某些异常现象,如染色体丢失,多极纺锤体形成,以及第二次卵裂时受精卵的两分裂球的非同步性分裂等。我们初步认为,雌核发育银鲫的染色体和两性融合生殖的鱼类染色体之间存在某种不相容性,表现为彼此间的互相排斥。出现这种现象可能和银鲫染色体组的雌核发育遗传背景有关。     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Ploidy of bovine nuclear transfer blastocysts reconstructed using in vitro produced blastomere donors

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.     

Triploidy--the breakdown of monogamy between sperm and egg

The advent of assisted reproductive technology (ART) has taught us a great deal about human fertilization patterns. Thirty years of experience with IVF and cultivation of early embryos has provided a unique view into the mechanisms of normal and aberrant human fertilization. Here we review the different types of triploidy following conventional in vitro fertilization and intracytoplasmic sperm injection, as well as the mechanisms giving rise to digynic and dispermic fertilization. Additionally, the role of the centrosome in triploidy, the genetic analysis of triploid embryos and the potential for therapeutic enucleation are explored. Lastly, we review our own clinical experience with human fertilization patterns following > 20,000 treatment cycles of assisted reproduction.     

Selective elimination of chromosomally unbalanced zygotes at the two-cell stage in the Chinese hamster

The gametic and zygotic selection of genome imbalance was investigated in the Chinese hamster by direct chromosome analyses of spermatocytes and preimplantation embryos from crosses between chromosomally normal females and males heterozygous for a reciprocal translocation, T(2;10)3Idr, abbreviated here as T3. The karyotypes and the frequencies of embryos observed at the first cleavage in the cross +/+female X T3/+male were consistent with those expected from MII scoring in male T3 heterozygotes. Therefore, it was concluded that there was neither gametic selection against genome imbalance nor zygotic selection from fertilization until the first cleavage metaphase. However, 9.1-10.8% of embryos were arrested at the two-cell stage, and karyotypes of these embryos were confirmed as 22(2,10,10,10(2)), 21(2,10,10), and 21(2,10,10(2)). The common abnormality of these embryos was partial monosomy of chromosome 2. Among day 4 embryos, some chromosomally unbalanced embryos, mainly with a deficiency of other segments of chromosomes 2 and 10, had fewer blastomeres than chromosomally balanced embryos. This finding suggests that cleavage of these embryos had been retarded by day 4 of gestation.     

Chromosome variation in the embryos of domestic animals

Chromosome abnormalities in the embryos of domestic animals are mostly eliminated during development. De novo chromosome abnormalities in the embryos of domestic animals have been detected in a larger proportion of embryos produced by in vitro fertilization and somatic cell nuclear transfer than in those produced by natural mating or artificial insemination. The increased incidence of abnormalities in embryos produced in vitro provides evidence for an influence of the embryo production procedures on chromosome stability. Research strategies involving cytogenetics, molecular biology and reproductive biotechnologies hold the promise of yielding insight into the mechanisms underlying chromosome instability in embryos and the impact of the in vitro environment on the chromosome make-up of embryos.     

Variable expressivity of the tumour suppressor protein TRP53 in cryopreserved human blastocysts

In a mouse model, in vitro fertilization or extended embryo culture leads to the increased expression of TRP53 in susceptible embryos. Ablation of the TRP53 gene improved embryo viability indicating that increased expression of TRP53 is a cause of the reduction of embryo viability resulting from in vitro fertilization or embryo culture. This study investigates the status of TRP53 expression in human embryos produced by intracytoplasmic sperm injection. Following fertilization, embryos were cultured for 96 h and then cryopreserved. Immediately upon thawing they were fixed in formaldehyde and subjected to immunostaining for TRP53. Staining was visualized by confocal microscopy. Negative controls were incubated with isotype control immunoglobulin and showed negligible staining. All embryos showed TRP53 staining above negative controls. TRP53 staining was heterogenous within and between embryos. An embryo that showed retarded development showed high levels of TRP53 expression. A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts. Most TRP53 staining was in the region of the nucleus. Morphologically normal blastocysts tended to show little nuclear accumulation of stain. However, some cells within these embryos had high levels of nuclear TRP53 expression. The results show that embryos have varying sensitivity to the stresses of production and culture in vitro, and this resulted in variable expressivity of TRP53.     

Mouse oocytes and early embryos express multiple histone H1 subtypes

Oocytes and embryos of many species, including mammals, contain a unique linker (H1) histone, termed H1oo in mammals. It is uncertain, however, whether other H1 histones also contribute to the linker histone complement of these cells. Using immunofluorescence and radiolabeling, we have examined whether histone H10, which frequently accumulates in the chromatin of nondividing cells, and the somatic subtypes of H1 are present in mouse oocytes and early embryos. We report that oocytes and embryos contain mRNA encoding H10. A polymerase chain reaction-based test indicated that the poly(A) tail did not lengthen during meiotic maturation, although it did so beginning at the four-cell stage. Antibodies raised against histone H10 stained the nucleus of wild-type prophase-arrested oocytes but not of mice lacking the H10 gene. Following fertilization, H10 was detected in the nuclei of two-cell embryos and less strongly at the four-cell stage. No signal was detected in H10 -/- embryos. Radiolabeling revealed that species comigrating with the somatic H1 subtypes H1a and H1c were synthesized in maturing oocytes and in one- and two-cell embryos. Beginning at the four-cell stage in both wild-type and H10 -/- embryos, species comigrating with subtypes H1b, H1d, and H1e were additionally synthesized. These results establish that histone H10 constitutes a portion of the linker histone complement in oocytes and early embryos and that changes in the pattern of somatic H1 synthesis occur during early embryonic development. Taken together with previous results, these findings suggest that multiple H1 subtypes are present on oocyte chromatin and that following fertilization changes in the histone H1 complement accompany the establishment of regulated embryonic gene expression.     

小鼠精子注入兔卵母细胞受精研究

The methods of intracytoplasmic sperm injection (ICSI) and subzonal injection (SUZI) were used to study heterologous fertilization and embryonic development between the mouse and the rabbit. Results were as follows: 1. The mouse sperm nuclei decondensed and formed pronuclei following microinjection into cytoplasm and perivitelline space (PVS) of rabbit oocytes; 2. The hybrid embryos developed to the stage of 8-cell when cultured in vitro; 3. The karyotype analysis showed a normal complement of rabbit oocyte and mouse sperm chromosomes in the 4-cell hybrid embryos; 4. The ultrastructure of 4-cell hybrid embryos was similar to that of normal 4-cell rabbit embryos; 5. The fertilization rate (32.4%) and cleavage rate (22.2%) when 5-10 mouse spermatozoa were injected were higher than those of injection of a single spermatozoon into PVS of the rabbit oocyte, but the difference was not significant (P > 0.05). The fertilization rate (42.3%) and cleavage rate (30.8%) in rabbit oocytes in vitro matured for 11-12 h were higher than those in the oocytes which were in vitro matured for 24-25 h following microinjection of 1-2 mouse spermatozoa into PVS, but the difference was not significant (P > 0.05).