Biomedical subjects

R Yanagimachi

Publications and source records attributed to R Yanagimachi.

At least 55 records · Page 3Linked to original sources

PubMed1999

Comparison of oocyte-activating agents for mouse cloning.

Since somatic cell components are unable to activate oocytes following injection or fusion, enucleated oocytes receiving adult somatic cells during the cloning process must be activated artificially for their development. We compared the efficiency of four types of oocyte-activating agents: strontium, ethanol, single electric pulse, and spermatozoa. Although strontium was the best in supporting preimplantation development of reconstructed mouse oocytes, there was no significant difference among the four agents with respect to the rate of postimplantation embryo development and the birth of live offspring.

Animals↗

PubMed1999

Status of genomic imprinting in mouse spermatids.

The advent of human round spermatid microinjection (ROSI) into oocytes as a treatment for severe male infertility raises the question of whether spermatids have undergone all of the maturation processes necessary for normal development. It is particularly important to know whether spermatids have undergone correct genomic imprinting, which results in the parent-of-origin-specific expression of only one allele of a gene. We assessed the imprinting status of three maternally and three paternally expressed genes in interspecific hybrid embryos generated by injecting Mus castaneus spermatids into Mus musculus oocytes. We used the single nucleotide primer extension (SNuPE) assay to measure the relative expression of maternal and paternal alleles on the basis of sequence polymorphisms in the transcripts. Expression of imprinted genes in mouse embryos derived by ROSI did not differ from controls, indicating that paternal genes have undergone proper imprinting by the round spermatid stage.

Animals↗

PubMed1999

Successful fertilization and pregnancy following ICSI and electrical oocyte activation.

In a total of 1048 intracytoplasmic sperm injection (ICSI) cycles, motile spermatozoa from four out of 424 patients (0.9%) failed to fertilize oocytes, despite an apparently successful ICSI procedure. No activation was observed in these injected oocytes. The spermatozoa from three of the four patients were injected into unfertilized mouse oocytes by ICSI (mouse test) to evaluate their oocyte activating ability. The oocyte activation rate of the spermatozoa of patients A, B, and C in the mouse test was 46, 100, and 86% respectively (control: 100%). Simultaneous injection of two spermatozoa from patient A into the mouse oocytes increased the oocyte activating rate to 89% (sham control: 29%). 100% fertilization rates were obtained for patients A and B by combining ICSI and electrical stimulation, and this resulted in pregnancy and the birth of healthy twins for the partner of patient A. Thus, it is considered that the spermatozoa of these patients are not lacking sperm factors but that the activity of these factors is depressed. The combination of ICSI and electrical stimulation is effective in these cases.

Animals↗

PubMed1999

An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development.

We have been interested in determining the minimally required elements in the sperm head that are necessary in order for the paternal genome to participate in embryogenesis. We used an ionic detergent, mixed alkyltrimethylammonium bromide (ATAB), plus dithiothreitol (DTT) to remove the acrosome and almost all of the perinuclear theca, leaving only the sperm nucleus morphologically intact. We also tested the stability of the sperm nuclear matrix by the ability to form nuclear halos. Sperm nuclei washed in freshly prepared 0.5% ATAB + 2 mM DTT completely decondensed when extracted with salt, but nuclei washed in the same buffer that was 1 wk old, and then extracted with salt, produced nuclear halos, indicating stable nuclear matrices. When we treated sperm heads with freshly prepared ATAB+DTT and injected them into oocytes, none of the oocytes developed into live offspring. In contrast, sperm heads treated in the same way but with 1-wk-old ATAB+DTT solution could support development of about 30% of the oocytes to live offspring. Electron microscopy demonstrated that most of the perinuclear theca had been removed in both cases. These data suggest that at least in the mouse, the only component of the spermatozoa that is crucial for participation in embryologic development is the sperm nucleus with a stable nuclear matrix.

Animals↗

PubMed1999

A novel trans-complementation assay suggests full mammalian oocyte activation is coordinately initiated by multiple, submembrane sperm components.

To initiate normal embryonic development, an egg must receive a signal to become activated at fertilization. We here report that the ability of demembranated sperm heads to activate is abolished after incubation over the range 20-44 degreesC and is sensitive to reducing agents. On the basis of this observation, we have developed a microinjection-based, trans-complementation assay in order to dissect the heat-inactivated sperm-borne oocyte-activating factor(s) (SOAF). We demonstrate that the failure of heat-inactivated sperm heads to activate an egg is rescued by coinjection with dithiothreitol-solubilized SOAF from demembranated sperm heads. The solubilized SOAF (SOAFs) is trypsin sensitive and is liberated from demembranated heads in a temperature-dependent manner that inversely correlates with the ability of sperm heads to activate. This argues that SOAFs is a proteinaceous molecular species required to initiate activation. Injection of oocytes with mouse or hamster sperm cytosolic factors, but not SOAFs alone, induced resumption of meiosis, further suggesting that these cytosolic factors and SOAF are distinct. Collectively, these data strongly suggest that full mammalian oocyte activation is initiated by the coordinated action of one or more heat-sensitive protein constituents of the perinuclear matrix and at least one heat-stable submembrane component.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules.

For many infertile couples, intracytoplasmic germ cell/spermatozoon injection into unfertilized eggs may be their only hope for producing their own biological children. Thus far, success with injection of pre-spermatozoan germ cells such as round spermatids has not been as great as that of spermatozoon injection. This could be due in part to the difficulty of identifying younger (less mature) male germ cells in testicular biopsy dispersions. To improve the identification of various types of live, dispersed, human testicular cells in vitro, a comparative study of the morphological characteristics of human spermatogenic germ cells in vitro or in situ within seminiferous tubules was conducted. Live human testicular tissue was obtained from an organ-donating, brain-dead person with a high density of various germ cells. A cell suspension was obtained by enzymatic digestion, and cells were cultured for 3 days in an excessive volume (100-fold medium:cells; v:v) of HEPES-TC 199 medium at 5 degrees C and observed live with Nomarski optics (interference-contrast microscopy). For comparative purposes, testes from ten men obtained at autopsy were fixed, embedded in epoxy resin, sectioned at 20 microm, and observed unstained by Nomarski optics. This approach allowed comparison of morphological characteristics of individual germ cells seen in vitro or in situ in the human testis. In both live and fixed preparations from control men with varied daily sperm production rates, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells. The size, shape, and chromatin pattern of nuclei, and the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece of germ cells are characteristically seen in live cells in vitro and in those cells observed in the fixed seminiferous tubules. Hence, this comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells, to be used by scientists and technical staff in infertility clinics when selecting specific germ cells from a testicular aspirate or enzymatically digested biopsy.

Adult↗

PubMed1999

Fertility of mouse spermatozoa retrieved from cadavers and maintained at 4 degrees C.

After male animals die, the spermatozoa within the testis and epididymis eventually disintegrate. In this study, the motility, viability and fertility of mouse spermatozoa were examined after retrieval from the epididymis at various days after death. Cadavers were maintained in a refrigerator at 4 degrees C. About 30% of the spermatozoa collected 10 days after death were viable, but they had limited ability to fertilize oocytes in vitro. However, when the spermatozoa were injected into oocytes, the fertilization rate was over 80%. Normal live fetuses were even obtained using immotile spermatozoa retrieved 20 days after death. Therefore, when valuable male animals die unexpectedly and sperm cryopreservation is not possible immediately, temporal storage of cadavers (or epididymis and vas deferens) at 4 degrees C in a regular refrigerator followed by intracytoplasmic sperm injection may help to preserve the genome of individuals. This procedure could be particularly important in endangered species.

Animals↗

PubMed1998

Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.

Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.

Animals↗

PubMed1998

Decondensation of the mouse sperm nucleus within the interphase nucleus.

Sperm nuclei incorporated into the cytoplasm (ooplasm) of fertilised mouse eggs at the pronuclear stage remain condensed, whereas those injected into male or female pronuclei decondense. Similarly, sperm nuclei injected into germinal vesicles of immature oocytes or the nuclei of 2-cell embryos decondense, while those entering the cytoplasm of these oocytes/embryos do not. These facts seem to suggest that factors necessary for the decondensation of sperm nucleus are present in interphase nuclei and are released into the ooplasm during nuclear envelope breakdown. Nucleoplasmin, which is synthesised in the cytoplasm and accumulated within the nucleus, is likely a major candidate for these factors.

Animals↗

PubMed1998

Intracytoplasmic sperm injection in the rat.

We applied intracytoplasmic sperm injection (ICSI) to the rat comparing three different sperm injection techniques: conventional setup with a sharp needle bearing a spike (method 1), combination of partial zona dissection (PZD) needle and blunt pipette (method 2) and piezo-injection using a blunt pipette (method 3). We also investigated the timing of sperm pronuclear formation after injection. Survival rates after injection were 8%, 24% and 71% for the methods 1, 2 and 3, respectively. All surviving oocytes formed pronuclei by about 6 h after injection. Although the survival and activation rates following sperm injection using piezo-injection were high, the incidence of normal fertilisation, as evidenced by second polar body extrusion and formation of two pronuclei, was only 10%. The vast majority of the zygotes were multinucleated, although most of them subsequently underwent cleavage. Fixation and staining of injected oocytes at different times after injection revealed that replacement of sperm nuclear protamines by histones takes place by 15 min after injection, sperm head swelling occurs within 0.5-1 h after injection and pronuclei become fully developed by 7 h after injection. Although the rate of normal fertilisation in the rat following ICSI was low under the present experimental conditions, the results indicated that direct ICSI using a piezo-driven pipette would be a potentially valuable method of producing rat offspring.

Animals↗

PubMed1998

Fate of microinjected spermatid mitochondria in the mouse oocyte and embryo.

Mouse round spermatids labelled with MitoTracker were microinjected into Sr(2+)-activated mouse oocytes. The labelled mitochondria were tracked up to the morula/blastocyst stage using fluorescence microscopy. The overall incidence of embryos with labelled mitochondria fell from 80% in the 1-cell zygote to 25% in 2-cell, 9% in 4-cell and approximately 1% in 8-cell or later stages. Thus it appears that almost all round spermatid mitochondria finally disappear from embryos during the 4-cell to 8-cell transition, as happens for mature spermatozoa (Cummins et al. Zygote 1997, 5:301-8). The spermatid mitochondria remained tightly bound together during this process. In contrast, labelled primary spermatocyte and cumulus mitochondria dispersed rapidly throughout the oocyte cytoplasm within 3 h. We hypothesise that spermatid mitochondria may be bound together by cytoskeletal elements produced in the early haploid spermatid. These elements, together with terminal differentiation of the sperm mitochondria, may be central to the processes by which the embryo 'recognises' the sperm mitochondria and inhibits inheritance of paternal mitochondrial DNA. These results suggest that round spermatid injection for infertile men will not pose a significant risk to offspring by transmitting abnormal mitochondrial genomes.

Animals↗

PubMed1998

Can alcohol retain the reproductive and genetic potential of sperm nuclei? Chromosome analysis of mouse spermatozoa stored in alcohol.

Alcohol is known to preserve genomic DNA and the primary structure of sperm protamines. To determine whether alcohol can retain the genetic and reproductive potential of mammalian sperm nuclei, mature mouse spermatozoa were stored in 70% ethanol or propanol for up to 2 months before injection into oocytes. Live offspring were obtained after injection of spermatozoa stored in 70% ethanol for 1 day at -20 degrees C. About 20% of the spermatozoa stored under this condition had normal chromosomes. The remaining 80% of spermatozoa and all the spermatozoa stored in 70% ethanol for 2 months had structurally aberrant chromosomes, and none could support the development of normal embryos. High concentrations of alcohol do not alter the primary structure of either DNA or small-molecular-weight protamines. However, alcohol may modify protamine-protamine or protamine-DNA interactions in a manner that results in the induction of DNA strand breaks during sperm chromatin decondensation within the oocyte. The limited success in obtaining normal offspring with ethanol-stored spermatozoa is encouraging. It may be possible to overcome these problems and develop a simple method for preserving mammalian spermatozoa without freezing.

Animals↗

PubMed1998

Fertilisability and developmental ability of mouse oocytes with reduced amounts of cytoplasm.

To determine the minimal amount of cytoplasm necessary to support normal development of mouse oocytes, mature unfertilised oocytes were reduced in size into approximately 1/2, 1/4 and 1/8 by removing the cytoplasm, then inseminated. More than 80% of 1/2-size oocyte were fertilised normally and almost all fertilised eggs developed to blastocysts. When transferred to foster females, 31% of the blastocysts developed into normal offspring. In contrast, 1/4- and 1/8-size oocytes, although they were penetrable by spermatozoa and extruded the second polar bodies, could not reach the 2-cell stage. In these oocytes, sperm nuclei did not develop into full-sized pronuclei. These results suggest that an oocyte can develop to full term after losing about half its cytoplasm, but not more.

Animals↗

PubMed1998

Comparison of the media for isolation and storage of round spermatid nuclei before intracytoplasmic injection.

PURPOSE: This study investigated whether K(+)-rich medium is better than pure NaCl solution or Na(+)-rich cell culture medium for handling round spermatid nuclei prior to injection into oocytes (ROSNI). METHODS: Round spermatids of the mouse were isolated and stored in isotonic NaCl, a cell culture medium (CZB), or a nucleus isolation medium (NIM) before injection into oocytes. The rates of normal fertilization, embryonic development in vitro, and birth of normal offspring after transfer of embryos to foster mothers were determined. RESULTS: In vitro development of ROSNI-produced zygotes to blastocysts was the same when "naked" spermatid nuclei were exposed briefly to three media. However, a long (60-min) exposure of the nuclei to Na(+)-rich medium was detrimental. In K(+)-rich NIM "naked" spermatid nuclei best retained their ability to participate in normal embryonic development. CONCLUSION: NIM was better than Na(+)-rich medium for retaining isolated spermatids competent to participate in normal embryonic development.

Animals↗

PubMed1998

Development of normal mice from oocytes injected with freeze-dried spermatozoa.

Freeze-dried mouse spermatozoa are all motionless and dead in the conventional sense. When injected into oocytes, however, their nuclei can support normal embryonic development even after three month preservation in a dried state. Although the freeze-drying protocol reported here will need further improvement, the results suggest that it may be possible to store the male genomes at room temperature.

Animal Husbandry↗

PubMed1998

Intracytoplasmic sperm injection experiments using the mouse as a model.

Due to the existence of ample background information on its reproduction, embryology and genetics, the mouse is potentially an excellent animal model for intracytoplasmic sperm injection (ICSI). Normal fertile mouse offspring have been obtained by ICSI using not only mature (epididymal) and immature (testicular) spermatozoa, but also round spermatids and secondary spermatocytes. This suggests that genomic imprinting of male germ cells is complete before spermiogenesis. Mature mouse spermatozoa carry one or more factors that activate oocytes. This sperm-borne oocyte-activating factor is present in testicular spermatozoa, but not in round spermatids. Thus, at least in the mouse, it seems to appear (or become active) during spermiogenesis. Part of the factor seems to be associated with the perinuclear materials because, when freed from plasma and acrosomal membranes as well as all acrosome components, spermatozoa remain fully capable of activating oocytes by ICSI. Spermatozoa with grossly misshapen heads (e.g. those from the BALB/c mouse) are unable to fertilize oocytes under ordinary in-vivo and in-vitro conditions. However, by ICSI they can fertilize the oocytes, and the zygotes develop into fertile offspring. Inherently poorly motile spermatozoa (of male mice carrying two t haplotypes) are unable to fertilize, but through ICSI they can participate in normal fertilization and embryonic development. Examination of human sperm chromosomes after sperm injection into mouse oocytes revealed that spermatozoa with abnormal head morphology have a significantly higher incidence of chromosome abnormality than those with normal heads, yet the majority of the abnormal spermatozoa have normal chromosomal constitutions. These findings suggest that spermatozoa with aberrant morphology and/or motility are not necessarily genomically abnormal.

Animals↗

PubMed1998

Mouse primary spermatocytes can complete two meiotic divisions within the oocyte cytoplasm.

This study was undertaken to determine whether primary spermatocyte nuclei can complete meiosis after transfer into maturing or mature oocytes and can participate in normal embryogenesis. When injected into maturing mouse oocytes at prometaphase of the first meiotic division, spermatocyte chromosomes became arranged on a first meiotic metaphase (Met-I) spindle. Thus, oocytes contained two sets of Met-I chromosomes. When these oocytes were matured in vitro and artificially activated, pronuclei and polar bodies of both maternal and paternal origin were formed, and zygotes began development. When single spermatocyte nuclei were injected into fully mature oocytes at metaphase of the second meiosis (Met-II), the spermatocyte nuclei transformed into a Met-I configuration, resulting in the formation of oocytes with both maternal (Met-II) and paternal (Met-I) chromosome complements. After activation of these oocytes, half of each chromosome set was separated into polar bodies. Transfer of nuclei from polar bodies of "paternal" origin into other Met-II oocytes resulted in the formation of oocytes with two sets of Met-II chromosomes, one maternal and one paternal in origin. When activated, two pronuclei and two polar bodies were formed and zygotes began development. It is concluded that nuclei of primary spermatocytes are able to undergo two successive meiotic divisions within oocyte cytoplasm. Thus, factors that drive chromosome condensation, organization of metaphase, and chromosome separation at anaphase in oocytes can drive these same maturation processes in primary spermatocyte nuclei. When a total of 258 two-cell embryos were transferred to foster mothers, only two live pups were born to two mothers. One died shortly after birth and the other 3 wk after birth. The reasons for poor embryonic and neonatal development remain to be determined.

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