Morphological analysis of the human sperm tail during the early post-fertilization stage.

After fertilization, the human sperm tail lost its fibrous sheath at the pronuclear stage to become coiled and then attached to one of the first mitotic spindle poles. The tip of the sperm tail was branched and its degree did not change from the pronuclear stage to the second mitosis.

Transcriptomic signatures in trophectoderm and inner cell mass of human blastocysts classified according to developmental potential, maternal age and morphology.

Selection of high-quality embryos is important to achieve successful pregnancy in assisted reproductive technology (ART). Recently, it has been debated whether RNA-sequencing (RNA-Seq) should be applied to ART to predict embryo quality. However, information on genes that can serve as markers for pregnant expectancy is limited. Furthermore, there is no information on which transcriptome of trophectoderm (TE) or inner cell mass (ICM) is more highly correlated with pregnant expectancy. Here, we performed RNA-Seq analysis of TE and ICM of human blastocysts, the pregnancy expectation of which was retrospectively determined using the clinical outcomes of 1,890 cases of frozen-thawed blastocyst transfer. We identified genes that were correlated with the expected pregnancy rate in ICM and TE, respectively, with a larger number of genes identified in TE than in ICM. Downregulated genes in the TE of blastocysts that were estimated to have lower expectation of pregnancy included tight junction-related genes such as CXADR and ATP1B1, which have been implicated in peri-implantation development. Moreover, we identified dozens of differentially expressed genes by regrouping the blastocysts based on the maternal age and the Gardner score. Additionally, we showed that aneuploidy estimation using RNA-Seq datasets does not correlate with pregnancy expectation. Thus, our study provides an expanded list of candidate genes for the prediction of pregnancy in human blastocyst embryos.

First mitotic spindle formation is led by sperm centrosome-dependent MTOCs in humans.

Unlike in mice, multinucleated blastomeres appear at a high frequency in the two-cell-stage embryos in humans. In this Point of View article, we demonstrate that the first mitotic spindle formation led by sperm centrosome-dependent microtubule organizing centers may cause a high incidence of zygotic division errors using human tripronuclear zygotes.

Assessment of developmental potential of human single pronucleated zygotes derived from conventional in vitro fertilization.

PURPOSE: The aim of this study was to non-invasively validate the developmental potential of human single pronucleated (1PN) zygotes derived from conventional in vitro fertilization (c-IVF) at the zygote stage. METHODS: Fifty 1PN zygotes derived from 45 patients undergoing c-IVF were used. Immunohistochemistry and fluorescence live cell imaging were used to confirm normal chromosome segregation during the first mitosis. The usefulness of measuring pronuclear diameter was assessed on the basis of the presence or absence of a proper first cleavage and validated by subsequent development. RESULTS: Although approximately 80% (15/19) of 1PN zygotes contained a diploid genome, immunohistochemistry revealed an unequal distribution of paternal and maternal genomes at the first mitosis. Fluorescence live imaging revealed that 73% (8/11) of 1PN zygotes formed a functional mitotic spindle at the first mitosis resulting from diploid genomes, with 25% (2/8) of these forming a tripolar spindle. 1PN zygotes in which the pronucleus disappeared and that subsequently underwent cleavage had a pronuclear diameter ≥ 32.2 µm. The selection of 1PN zygotes based on pronuclear diameter resulted in zygotes that all formed mitotic spindles with poles during cleavage. Furthermore, 63% (5/8) of these zygotes reached the blastocyst stage. CONCLUSIONS: This study demonstrates the usefulness of a non-invasive assessment of 1PN zygotes derived from c-IVF as an indicator of developmental potential. Furthermore, diploid 1PN zygotes do not always exhibit normal chromosome segregation at the first mitosis. A pronuclear diameter ≥ 32.2 µm just before PN breakdown might be a useful criterion to assess 1PN zygotes that are capable of further development.

Diagnosis of abnormal human fertilization status based on pronuclear origin and/or centrosome number.

PURPOSE: Normally fertilized zygotes generally show two pronuclei (2PN) and the extrusion of the second polar body. Conventional in vitro fertilization (c-IVF) and intracytoplasmic sperm injection (ICSI) often result in abnormal monopronuclear (1PN), tripronuclear (3PN), or other polypronuclear zygotes. In this study, we performed combined analyses of the methylation status of pronuclei (PN) and the number of centrosomes, to reveal the abnormal fertilization status in human zygotes. METHOD: We used differences in DNA methylation status (5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)) to discriminate between male and female PN in human zygotes. These results were also used to analyze the centrosome number to indicate how many sperm entered into the oocyte. RESULT: Immunofluorescent analysis shows that all of the normal 2PN zygotes had one 5mC/5hmC double-positive PN and one 5mC-positive PN, whereas a parthenogenetically activated oocyte had only 5mC staining of the PN. All of the zygotes derived from ICSI (1PN, 3PN) had two centrosomes as did all of the 2PN zygotes derived from c-IVF. Of the 1PN zygotes derived from c-IVF, more than 50 % had staining for both 5mC and 5hmC in a single PN, and one or two centrosomes, indicating fertilization by a single sperm. Meanwhile, most of 3PN zygotes derived from c-IVF had a 5mC-positive PN and two 5mC/5hmC double-positive PNs, and had four or five centrosomes, suggesting polyspermy. CONCLUSIONS: We have established a reliable method to identify the PN origin based on the epigenetic status of the genome and have complemented these results by counting the centrosomes of zygotes.

Analysis of compaction initiation in human embryos by using time-lapse cinematography.

PURPOSE: To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. METHODS: One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. RESULTS: Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. CONCLUSIONS: The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis).

Possible mechanism of polyspermy block in human oocytes observed by time-lapse cinematography.

PURPOSE: To analyze the fertilization process related to polyspermy block in human oocytes using an in vitro culturing system for time-lapse cinematography. METHODS: We had 122 oocytes donated for this study from couples that provided informed consent. We recorded human oocytes at 2,000 to 2,800 frames every 10 s during the fertilization process and thereafter every 2 min using a new in vitro culture system originally developed by the authors for time-lapse cinematography. We displayed 30 frames per second for analysis of the polyspermy block during fertilization. RESULTS: Three oocytes showed the leading and following sperm within the zona pellucida in the same microscopic field. The dynamic images obtained during the fertilization process using this new system revealed that once a leading sperm penetrated the zona pellucida and attached to the oocyte membrane, a following sperm was arrested from further penetration into the zona pellucida within 10 s. CONCLUSIONS: The present results strongly suggest the existence of a novel mechanism of polyspermy block that takes place at the zona pellucida immediately after fertilization. These findings are clearly different from previous mechanisms describing polyspermy block as the oocyte membrane block to sperm penetration and the zona reaction. The finding presented herein thus represents a novel discovery about the highly complicated polyspermy block mechanism occurring in human oocytes.

Enhanced apoptosis during early neuronal differentiation in mouse ES cells with autosomal imbalance.

Although particular chromosomal syndromes are phenotypically and clinically distinct, the majority of individuals with autosomal imbalance, such as aneuploidy, manifest mental retardation. A common abnormal phenotype of Down syndrome (DS), the most prevalent autosomal aneuploidy, shows a reduction in both the number and the density of neurons in the brain. As a DS model, we have recently created chimeric mice from ES cells containing a single human chromosome 21. The mice mimicked the characteristic phenotypic features of DS, and ES cells showed a higher incidence of apoptosis during early neuronal differentiation in vitro. In this study, we examined the induction of anomalous early neural development by aneuploidy in mouse ES cells by transferring various human chromosomes or additional mouse chromosomes. Results showed an elevated incidence of apoptosis in all autosome-aneuploid clones examined during early neuronal differentiation in vitro. Further, cDNA microarray analysis revealed a common cluster of down-regulated genes, of which eight known genes are related to cell proliferation, neurite outgrowth and differentiation. Importantly, targeting of these genes by siRNA knockdown in normal mouse ES cells led to enhanced apoptosis during early neuronal differentiation. These findings strongly suggest that autosomal imbalance is associated with general neuronal loss through a common molecular mechanism for apoptosis.

Dlx5, the mouse homologue of the human-imprinted DLX5 gene, is biallelically expressed in the mouse brain.

The mouse Dlx5 gene encodes a distal-less-related DNA-binding homeobox protein first expressed during early embryonic development in anterior regions of mouse embryo and is located on chromosome 6, which is the syntenic region to the human chromosome 7q21-q31 imprinting cluster. Recently, its human homologue, DLX5, was identified to be imprinted and maternally expressed, at least in normal human lymphoblasts and in brain tissues. In our study, we analyzed the imprinting status of mouse Dlx5 by RT-PCR, first in the F1 of a reciprocal cross between two different mouse strains, and second in heterozygous Dlx5 mutant mice. Both approaches revealed that mouse Dlx5 followed a biallelic pattern of expression in brain tissue and in testis. Our findings suggest that the Dlx5 gene escapes genomic imprinting, at least in mice of certain genetic backgrounds.

Human chromosome 21q22.2-qter carries a gene(s) responsible for downregulation of mlc2a and PEBP in Down syndrome model mice.

Congenital heart disease (CHD) is a major clinical manifestation of Down syndrome (DS). We recently showed that chimeric mice containing a human chromosome 21 (Chr 21) exhibited phenotypic traits of DS, including CHD. Our previous study showed that myosin light chain-2a (mlc2a) expression was reduced in the hearts of chimeric mice and DS patients. We found that phosphatidylethanolamine binding protein (PEBP) was also downregulated in Chr 21 chimeras in this study. As mlc2a is involved in heart morphogenesis, and PEBP controls the proliferation and differentiation of different cell types, these genes are candidates for involvement in DS-CHD. The DS-CHD candidate region has been suggested to span between PFKL and D21S3, which is the STS marker near the ETS2 loci. To identify gene(s) or a gene cluster on Chr 21 responsible for the downregulation of mlc2a and PEBP, we fragmented Chr 21 at the EST2 loci, by telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. The modified Chr 21 was transferred to mouse ES cells by microcell-mediated chromosome transfer (MMCT), via CHO cells. We used ES cell lines retaining the Chr 21 truncated at the ETS2 locus (Chr 21E) to produce chimeric mice and compared overall protein expression patterns in hearts of the chimeras containing the intact and the fragmented Chr 21 by two-dimensional electrophoresis. While mouse mlc2a and PEBP expression was downregulated in the chimeras containing the intact Chr 21, the expression was not affected in the Chr 21E chimeras. Therefore, we suggest that Chr 21 gene(s) distal from the ETS2 locus reduce mouse mlc2a and PEBP expression in DS model mice and DS. Thus, this chromosome engineering technology is a useful tool for identification or mapping of genes that contribute to the DS phenotypes.